Vol 5

2014
Infrared Reference Spectra

V-S2 Infrared Reference Spectra 2014
Preparation of Infrared Reference Spectra
AH spectra presented in this section were recorded using either a

Perkin-Elmer model 682 dispersive infrared spectrophotometer, a
Perkin Elmer model 16PC Fourier transform infrared
spectrophotometer or a Perkin Elmer model Spectrum 100 Fourier
transform infrared spectrophotometer.
Pressed discs, 13 mm in diameter, were prepared using
potassium bromide or potassium chloride. Liquid paraffin muHs
and thin films were prepared between potassium bromide pIates,
and gas and solution spectra were prepared using ceHs with
potassium bromide windows. Solution spectra were prepared
against a solvent reference and aH other spectra were recorded
against airo Attentuated Total Refiectance (ATR) spectra were
recorded by pressing the sample against a horizontal diamond
ATR crystal.
For solution spectra the regions of the spectrum within which
the solvent shows strong absorption should be disregarded. Solvent
'cut-offs' in the reference spectra may be recorded as horizontal
straight lines or may appear as blank regions on the spectrum.
2014 Infrared Reference Spectra V -S3
Polystyrene Instrument: Dispersive Phase: Thin Film Thickness : O.038mm

100.0 .
80
60
40 L. . .
~
Q)
ü 20 .
e
ro
::=
'E
(J)
e
ro
.= 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm" )
60
¡
+- ----..
g 20 · t~ ________ '·_··~~ __·~"··~ 1

ro
::=
'E
(J)
e
~
f- 0.0 -j.......... _ ... _ .. __ . • ; . . . ' - '" ....\..1. ........ .. _j ............ _ ........ __ .. ~
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
V -S4 Infrared Reference Spectra 2014
Polystyrene Instrumenl: Fourier Transform Phase: Thin Film Thickness: O.038mm

100.0
80
60
40
cf!.
Cl)
'-' 20
e
ro
;::::
·E
en
e
~
r- 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-')
100.0 ..,..,_ __
I
80 :. ___ .
60 I
.~_
I
I
40 _______ _
I I
g 20 +______._
g
-E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Abacavir RS464 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100.0 _. _____ ~_ - - - - . - . - -- - ·----r-·--··--··---·,..
,
I
60 '_
I
i
í
40 J __
¡'5 20
e
ro
:::::
-E
en
e
ro I
.= 00 : . __. . --- -~--
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
2014 Infrared Reference Spectra V-SS
Acebutolol Hydrochloride RS380 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
80
60
I
40 J .. . }--
I
;f'!.
ID
u 20
e
:§
·E
m
e
ro
.= 0.0 -1 J
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Acenoco um arol RSOOJ Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
I
80
60
40 ~ ,,
4-
;f'!.
ID
u 20
e
:§
-E
m
e
,
I
ro
.= 0.0 1 ~,
I
.....
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Acctazolamide RS002 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40 ;
I
¡
I
1
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Acetylcysteíne RS003 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
;f2.
~
e
ro
::=
'E
en
e
ro
t= O.OL
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-')
;f2.
Q)
(,) 20
e
ro
::=
'E
en
e
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Adrenalíne (Epínephríne) RS004 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 . ,......................................
80
60
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
2014 Infrared Reference Spectra V-S7
Alfuzosin RS446 Instrument: Fourier Transform Phase: Potassium Chloride Disc

100.0
60
40
20
0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Alimemazine RS005 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0. 1mm
100.0
80
60
40
~
Q)
o 20
e
ro
E
E
rJ)
e
ro
¡:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Alverine Citrate RS409 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
80
60
40
~ 20
e
ro
:::e
E
rJ)
e
ro
¡:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
v-ss Infrared Reference Spectra 2014
Amantadine RS006 Inslrument: Dispersive Phase: Polassium Bromide Disc
?F.
CD
o 20
e
,¡g
'E
(J)
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Aminoglutethimide RS403 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100.0
60
o~
CD
o 20
e
,¡g
'E
(J)
e
ro
~ 0,0 j
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Amiodarone RS008 Inslrument: Dispersive Phase: 15% w/v Solulion in Dichloromelhane Thickness : O.lmm
100,0
60
~ 20
e
,¡g
'E
(J)
e
ro
~ 0,0 _;_..__~. .f-.-..
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Amisulpride RS462 Instrumenl: Fourier Transform Phase: Attenuated Total Reflectance

100, O~;:;::;;::;::::::;.==::;:::::=
80
60
<f!.
u
Q)
20
e
g
'E
en
e
ro
.= 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400 ,0
Wavenumber (cm" )
Amoxicillin Sodium RSOIO Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~
Q)
u 20
e
ro
::::::
'E
en
e
~
f- 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Amoxicillin Trihydrate RSOll Instrumenl: Dispersive Phase : Potassium Bromide Disc

100.0 _,~_~~.,~,
80
40
2000,0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm" )
v-S 1O Infrared Reference Spectra 2014
Ampicillin Sodium RS366 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T " ............................... . ,•................................ .
60
o~
Q)
ü 20
e
ro
:;:::
'E
en
e
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ampicillin Trihydrate RS013 Instrument: Dispersive Phase: Potassium Bromide Disc
?f2.
Q)
ü 20
e
ro
:;:::
'E
en
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Amylmetacresol RS014 Instrument: Dispersive Phase : Thin Film

100.0 ., ......................... ................. ........,......•......... ........................... .. ,. . ..................................
~
~ 20
e
g
'E
en
e
~ 0.0 + ...........___ ~ _..f - ..•...._-_.-:--_.•- ......_....~ ...._ ..L
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2014 Infrared Reference Spectra V-S 11
Atenolol RS015 Instrument: Dispersive Phase: Potassium Bromide Disc
<f?
al
o 20
e
ro
::=
-E
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400_0
Wavenumber (cm-')
Azapropazone RSOl6 Instrument: Dispersive Phase: Potassium Bromide Disc
<f?
al 20
o
e
ro
::=
-E
(J)
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 _0
Wavenumber (cm-')
Anhydrous Azapropazone RS017 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60
40
2l 20
e
~
-E
(J)
e
~
f- o-o
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
v-S 12 Infrared Reference Spectra 2014
Azelastine Hydrochloride RSOl8 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
80
60
~
e
20 --_._ _-_._-,
.. -
... .. _-_..----
ro
:::::
·E
(f)
e
ro
t.':::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Beclometasone Dipropionate (1) RS020 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1 mm
<f?
ID
u 20
e
ro
:::::
-E
(f)
e
ro
t.':::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Beclometasone Dipropionate (2) RS379 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
80
60 + .......... .. . . . . ..
40
1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Beclometasone Dipropionate Monohydrate RS021 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc
100.0
80
60
<Ji!.
Q)
o 20
c:
ro
~
E
en
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Benorilate RS023 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100. O.'.. 0 •• ••• __ . . . . . . ........ . . . . , . . .. ,• • • •
60 + . .... . . . . . . . .
40
~
Q)
o 20
c:
ro
.~
E
en
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Benzatropine Mesilate RS026 Instrumenl: Dispersive Phase: Liquid Paraffin Mull

100.0
60
40
~ 20
c:
ro
:::::
'E
en
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
v -S 14 Infrared Reference Spectra 2014
Benzoic Acid RS025 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Benzydamioe Hydrochloride RS027 Instrument: Fourier Transform Phase: Potassium Ch loride Disc
100.0
60
40 t --
;
I
o~
Q)
o 20 j
e I
ro
:t='
-E
en
I
e
ro
.= 0.0 .1.
+-
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Benzyl Hydroxy benzoate RS028 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
80 t'"
¡
I
60 ¡
40 ~...--'~ ~.
~ - - --.-~ . ~--
<f!.
o
Q)
20 .... ~ ..1
e
ro
:t='
-E
en
e
ro
.= 0.0 .1. _ I
-\ ~--'(._.
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Betamethasone RS029 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
80
60
40
~
Q)
o 20
e
ro
:::::
·E
en
e
.=
ro
0.0 ).
i
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Bezafibrate RS419 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100.0
60
40
~
Q)
o 20
e
g
E
en
e
ro
.= 0.0 ¿.. 1
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Bicalutamide RS466 Inslrument: Fourier Transform Phase: Pol assium Bromide Disc
100.0
80
60
40 ~L.
o~
Q)
o 20
e
ro
:::::
·E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
v-S 16 Infrared Reference Spectra 201 4
Bleornyci n Sulfate RS367 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ..... .. .
"cf!.
w 20
u
e
ro
~(j)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Bretyliurn Tosilate RS030 Instrument: Fourier Transform Phase: Pota ssium Bromide Disc
100.0 .,.. . . ......... . . . . .
o~
w 20
u
e
ro
:::::
·E
(j)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Bronopol RS03 1 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60 .....
40
2l 20 + . . . . . . . . ----
e
ro
:::::
·E
(j)
e
ro
.= 0.0 1 ,
''"\-""----_.-
!
.... ?
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
2014 Infrared Reference Spectra V-S 1 7
Buclizine Hydrochloride RS032 Instrument: Dispersive Phase: Potassium Ch loride Disc

100.0 .¡
80
60
40 +---_________·_________+-____ H~
~
Q)
ü 20
e
ro
:::::
'E
(fl
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Bumetanide RS033 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 _
60
i
40 ·· ····· 1
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Bupivacaine RS034 Instrument: Dispersive Phase : Liquid Paraffin Mull

100.0 T ---
60
1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Busulfan RS035 Instrumenl: Dispersive Phase: Potassium Bromide Disc
40 L.
I
?ft.
Q)
o 20
e
ro
.",
'E rJ)
e
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Butyl Hydroxybenzoate RS036 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0 r - ----.-~---.----:- --
! .
80
60
40 +. I .... -...... "~'"f-''~'~'''~''' ... _- '1
o~
Q)
o 20
e
ro
.",
'E
rJ)
e
ro
.= í
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
100.0 r Calcium Folinate RS368 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc
I
60 ; . _______ _
~ 20 .¡. ___ ..•....__,. -1- --

e
ro
.",
'E
rJ)
e
~ 0.01 -y--'-'--
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm- 1)
Calcium Polystyrene Sulfonate RS037 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
80
60
40
;#.
Cll
o 20
c
ro
::=
-E
(J)
c
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Captopril RS038 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40 : ______ ___ _
;#.
Cll
o 20
c
ro
::=
-E
(J)
c
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Carbamazepine RS406 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
60
40
¡:¡ 20
c
ro
::=
-E
(J)
c
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Carbaryl RS039 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
40
o~
(1)
u 20
c:
ro
:::::
-E
en
c:
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Carbenoxolone RS041 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
~~y-,- _ _o - ~
40
o~
(1)
u 20
c:
ro
:::::
-E
en
c:
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Carbimazole RS042 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
60
40 -;______ ._ . . ._ ___
_.~ ~_~y J( ..
,I
.]
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Cefalexin RS049 Instrument: Fourier Transform Phase: Potassium Bromide Disc
~
o
ID
o 20
e
ro
::=
'E (J)
e
ro
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm- ) I
Cefotaxime Sodium RS044 Instrument: Dispersive Phase: Potassium Bromide Disc
o~
ID
o 20
e
~
E
(J)
e
ro
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm- I )
Cefoxitin Sodium RS045 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100,0
80
60
~ 20
e
:§
'E
(J)
e
~ 0.0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-I )
Cefradine RS050 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
I
60 ¡
40
<f?
Cll
u 20 _"~", " ~·,· m _,"h, ..
e
ro
::::::
·E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ceftriaxone Sodium RS046 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 T ·······r·····
i
2000.0 1800 1600 1400 1200 1000 800 400.0

Wavenumber (cm-')
Cefuroxime Axetil RS047 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
T·
1
80 ,
~._..;,...."~~_ .. _ . ~,
60
40
~ 20 1- - - ~ ... 1"".__ .
~
:::::: 1
·E
en
e
ro
.= 0.0 -1 {--
__ L_
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Cefuroxime Sodium RS048 Instrument: Fourier Transfonm Phase: Potassium Bromide Disc
100.0 "'r-
I
60 ¡
40 ~
,
~ I
(l)
u 20 j
e
ro
::=
·E
(J)
e
ro
.= 0.0 l
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- ) t
Celiprolol Hydrochloride RS420 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
T
80
60
I
1
40 . ~ _._.
J
I1
. . j .... ~
!
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Cetirizine Hydrochloride RS456 Instrument: Fourier Transform Phase: Potassium Btomide Disc
100.0
80
60 ______=. . . . .
40
~ 20
e
ro
::=
·E
(J)
e
ro
.= 0.0 j ----~
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-t )
Chlorhexidine RS449 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100. O.,....................................................·T··················································r .......-.........................................."............••.......•............•.•.- •.. c······································ -......•...,•.... - ...•...........•.........................., ..........
<f?
Q)
ü 20
e
ro
:;:::<
'E
en
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Chloroform RS053 Instrument: Dispersive Phase: 0.1mm Layer
40 . ¡-------.-.......... + ... ..__... ..._____. . . . . __ 1 ......... _.......•....... -.... + .... -._-.
<f?
Q)
ü 20
e
ro
:;:::<
'E
en
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Chloroquine RS054 Instrument: Dispersive Phase: 5% wlv Solution in Chloroform Thickness: 0.1mm
100.0 .,-.-....................-......................... . ...........-- ...... T'" ·.··.··-········.·-_··.·· ···.····T·····. ···· •.........••........ --.•- •.•.,..• .....•..
~ 20 +--·----···· ..·· ·~,, -·~~~-·_·-4H i--·--·-·-·-_·-·+~~~~_·--+-,,- --·- ·-----··r·.. --·-

e
:f1
'E
en
e
.=ro 0.0 +__________..__--+_._. __......_. __j-____ ._____._____+ ___... ___.. ___ .+____...___... . ______ 1
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Chloroxylenol RS055 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~
<1l
u 20
e
ro
E
Een
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Chlorpromazine RS056 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Th ickness: O.1mm
100.0
80
60
40
<f2.
u
<1l 20
e
g
E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 .0
Wavenumber (cm-1)
Chlorpropamide RS057 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
80
60
40
~ 20
e
ro
:l::'
-E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1)
V-S26 Infrared Reference Spectra 201 4
C hlortalidone RS058 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .
80
60
40 ~
I
e;e. I
Q)
ü 20 ,:
e I
ro
::=
'E
(J)
e
I
ro
t'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Choli ne Salicylate RS059 Instrument: Dispersive Phase: Thin Film

100.0
80
1
40 .Í"------
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
C holine T heophyllinate RS060 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
,
40 J
#-
~ 20 _j
I
¡¡j I
::=
'E
(J)
e
ro
t'= O.O . J.._ .......... _...••..•_ •...
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ciclosporin RS445 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
~
Q)
o 20
e
ro
:t:'
'E (J)
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Cimetidine RS061 Instrumenl: Dispersive Phase : Potassium Bromide Disc
<f2.
Q)
O 20
e
:§
'E
(J)
e
ro
f'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Cinnamic Acid RS062 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~ 20
e
ro
:E
E
(J)
e
ro
f'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
V-S28 Infrared Referen ce Spectra 201 4
Clarithro rnycin RS424 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 .
80
60
40
~ 20 t
·1(J)
c:
j
~ O.O !
2000.0 1800 1600 1400 1200 1000 600 400.0
Wavenumber (cm-1)
Clindarnycin Hydrochloride RS064 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 _ _ __ ~~~_ - --------r-- - - - ~ y ' - ' - - _ .. _ - - - - , - - - , - - - -
80 j
40
<f2.
25
c:
20 , ~
ro
::=
'E
(J)
e
~
r- 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Clioq uinol RS065 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T --'- - - - ,
80
60 ---~ ... -
40 _._-_._----,y,._.--" --- -
o - _.~- ,- --,------_.-
<f2.
al
u 20
c:
ro
::=
'E
(J)
c:
ro
~ 0.0 .;--_.. - .- •• A ... _ _ " _ _ _ _ _
2000.0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm-1)
Clobazam RS066 Instrumenl: Dispersive Phase: Potassium Bromide Disc
60
40
cft.
Q)
t.l 20
c::
ro
::::
'E
(f)
c::
ro
.= 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Clobetasol Propionate RS067 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100 ,O ,..---.-.-.....'-..,.-..--.--.. ~ . -..,'-..--..---.-...----c .....- ......._ ...-...... -_.
60 .} ......_ _ _ _,...__\
40 .....
~
o
Q)
t.l 20
c::
ro
::::
'E
(f)
c::
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
100.0 r"- Clofazimine RS068 Instrumenl: Dispersive Phase: Liquid Paraffin Mull
80
60
.. + --_.........., .........j..... -- ---_...... --,---+- ... --.... .'-' ....~, .... - ,

2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
CIomethiazole RS051 Instrument: Díspersive Phase: Thin Film

100.0
80
60
40
cf2.
Q)
o 20
e
ro
:::::
'E
UJ
c:
ro
,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
CIomethiazole Edisilate RS052 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0
80
60
40
cf2.
Q)
o 20 1
c:
ro
:::::
'E
UJ
c:
ro
,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Clomipramine Hydrochloride RS069 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
80
60
40
2:5 20 :
c:
-~
E
UJ
c:
ro
,:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Clonazepam RS432 Instrumenl: Fourier Transform Phase : Potassium Bromide Disc

100.0
80
60 ¡
40
~ 20
e
ro
:::::
"E
(f)
e
~ 0.0 j
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Clozapine RS444 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
r-
60
40
o~
ID 20
u
e
ro
:::::
"E
(f) ,
e
I
~ O.O·t 1"- ._~ .,
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Cocaine RS071 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100.0 _1 • .
80
60
40 .:_____ "
~ 20 .
e
ro
:::::
E
(f)
e
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 .0
Wavenumber (cm-')
V-S 32 Infrared Reference Spectra 201 4
Codeine RSOn Instrument: Dispersive Phase: Potassium Bromide Disc

100,0
40
u
Q)
20
e
ro
:1::'
-E
U)
e
ro
f'= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Colestyramine RS369 Instrument: Fourier Transform Phase: Potassium Bromid e Disc

100,0
60
~
Q)
u 20
e
ro
:1::'
-E
U)
e
ro
f'= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
1000 · · . . . .
Compressible Sugar RS401
r
Instrument: Fourier Transform Phase: Potassium Bromide Disc
80 .j:::::::::::=:::_.:::: _,-=~=--_"
.,-=:;
60
2.l 20
e
ro
:1::'
-E
U)
e
ro
f'= 0,0
2000_0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Cyclizine RS075 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0
80
60
Wavenumber (cm-')
Cyclizine Hydrochloride RS076 Instrument: Dispersive Phase: Liquid Paraffin Mull

100,0 1
60 ,
40 •• ,_ _
,
.v~., ~~,_ ,_. __ __ ,
I
O,0 i- .¡
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Cyclopenthiazide RS077 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0
80
60
~ 20
e
ro
::::
'E
(JJ
e
~
1- 0.0 .
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Cyclopentolate RS078 Instrument: Dispersive Phase: Thin Film

100.0
60
I
I
40
4 I
cF- I
I
Cl)
20 I
ü
e
ro
r
:::e I
'E
.:::
en
e
ro
0.0
2000.0
l. 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Cyclophosphamide RS079 Instrument: Dispersive Phase: 10% wlv Solution in Chloroform Thickness: 0.1mm
1000 .........................~~""'"
I
60 j'
I
40 i----. -----~--.
25
e
20
ro
:::e
'E
en
e
ro
.::: 0.0 . _______ o
_., . _- ----¡-...
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm")
Cyproheptadine RS080 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 .¡ ,
!
I
I
40 ~
,
I
';€
o
!
Cl)
ü
e
ro
20 lI
:::e I
'E
en
e
!
ro
.::: 0.0 --- -:
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Cyproterone Acetate RS395 Instrument: Fourier Transform Phase: Potassium Bromide Disc
~
Q)
u 20
e
ro
:::::
·E
(J)
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Cytarabine RS081 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
I
40 .~.
~ 20 +
~ 1
E
i
(J)
e
~ O
.ot. ...... _ . ~._ -_.-
i
4- · V~. _ _ · _,_,
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Dacarbazine RS082 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~
¡g 20
e
ro
:::::
·E
(J)
e
~ 0.0 ..;__
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Dantrolene Sodium RS422 Instrumen!: Fourier Transform Phase: Potassium Bromide Disc
100.0
80 ;
60
40
?,€
Q)
u 20
e
ro
!='
E
(J)
e
ro
r'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Dapsonc RS084 Instrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
"
40 -l
,,
8e 20
ro
!='
'E
(J)
e
ro
r'= 0.0 ~
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Desfcrrioxaminc Mesilatc RS086 Instrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 .
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
Desipramine Hydrochloride RS087 Inslrument: Dispersive Phase: Polassium Chloride Disc

100,0
60
40
~
CD
<..> 20
e
ro
::::::
'E
en
e
ro
t= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Desogestrel RS088 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100,0
~
o
CD
<..> 20
e
:§
'E
en
e
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm- ) I
Dexamethasone RS089 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100,Or.' ..........................................,..................................... . _. .. ,' .................................. _......... -,.........
60
40 +-~_.~ - - - t - - - -l
~ 20
e
ro
::::::
'E
en
e
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
DcxtJ"Omoramidc RS090 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
40 ·t····
o~
Q)
u 20 .~
I ,.¡
I
e
ro
:::::
'EU)
~
e
ro
0.0
2000.0
l.. . . 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dcxtropropoxyphcnc RS091 Instrument: Dispersive Phase: Thin Film

100.0
60 .
40 ; .1
I
~ 20 +__. ~ __~,....
e
ro
:::::
'E
U)
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dcxtropropoxyphcnc Napsilate RS092 Instrument: Dispersive Phase : Liquid Paraffin Mull

100.0
I
60
1 ¡
i
40 .t-_.- ._~, .
I
~ 20
e
:§
'E
U)
e
~ 0.0 '1"'- I - - - . - .-.-. -,,--
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Diamorphine HydrochlOJ'ide RS093 Inslrument: Dispersive Phase: Polassium Chloride Disc

100.0 .
60
40 v.- -----+---..--~-·--·-·----.--
. I
¡
,;
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Diazoxide RS094 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
60
40 .,
~
Q)
o 20
e
ro
:t:'
'E
(J)
e
ro
.= 0.0 +
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dibrompropamidine Isetionate RS095 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
80 .... .. .
60
40 ....
[I5 20
e
ro
:t:'
'E
(J)
e
ro
.= 0.0 ___ _
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Diclofenac RS096 Instrumenl: Fourier Transform Phase: Polassium Bromide Disc

100.0
60 " ................................
<f!.
Q)
o 20
e
ro
:::::
'E
(J)
e
~
r- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Diclofenac Diethylamine RS371 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc
100.0 .._..._.................... ...._ ·.....··--··-r· .... · .. ··· .. ·· .. ····· .. ·· .. ··.... · .,._-_ ..._ ..... .
60
o~
Q)
o 20
e
ro
:::::
'E
(J)
e
ro !
.= 0.0 ---J
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Dicycloverine Hydrochloride RS098 Inslrumenl: Dispersive Phase: Potassium Chloride Disc

100.0 . r-.~..._...._ ......_"'--_........_--,-_.._ - -.........>"-...- - - - . . . . . , . . . . . - - -
60
40 .'--- ---~
<f!.
C])
o 20
e
ro
:::::
'E
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Diethvlamine Salicylate RS099 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .
80
60
40
<f?
u
Q)
20
e
:§
'E
(/')
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Diethylcarbamazine RS411 Instrumenl: Dispersive Phase: Thin Film

100.0
<f?
Q)
u 20
e
ro
::::<
'E
en
e
.=ro 0.0 , .. ,
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Ditlucortolone Valerate RSIOO Instrumenl: Fourier Transform Phase: 5% w/v Solution in Dichloromethane Thickness: 0.1mm
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
Diflu nisal (form B) RSIOl Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
60 ,. .......... .
,
40
.- '1---"'--
o~
I,
Q)
() 20
e
ro
:::::
'EU)
e
~ 0.0 ~ .. _ " - "." -
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dihydrocodeioe RS 102 Instrument: Dispersive Phase: Thin Film

100.0 ."...................................r .........._•.~....., ........., ............., ...................,-.........., ..................., ...........',........, ........
80
40
~
o
Q)
() 20
e
ro
:::::
'EU)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Diloxanide Furoate RSI03 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .,...........................
60 .}-.................................. +. . . 1 .,
!,
,
_. -'~--~~-~ !
~ 20
e
ro
:::::
'E
U)
e
~ O.O+-________-+~..____.____r-_______ ._~~___.__..____ ¡__.________ . ___ . __~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Dimen hydrinate RS 104 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 -'~'T'-~"-""
60
40 . . __ ._----+---+-- _._-~ ...
I
·1··
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dimethyl Phthalate RSI05 Instrument: Dispersive Phase: Thin Film

100.0 .
60 ,..
¡
40
I
1
... 1'..........
?ft.
Q)
'-' 20
e
ro
:t:::
'E(f)
e
ro
.= 0.0 ... ~ .~' •.. _, . • ~v "-j"~" ~_._. __
___ . ...-- __4 _ _
" .... _~'; . ... ,"'(",., .,_ ..._----(
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dimeticone RS381 Instrument: Fourier Transform Phase: Thin Film

100.0 ~ _._---
1
i
I
80 .1
I
60 j.
40
?ft.
Q)
'-' 20
e
ro
:t:::
'E
(f)
e
~ 0.0 4-_ ...• ~_.,,-_.~_._- '.~ .. ,:.... ,~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Dinoprostone RS448 Instrument: Fourier Transform Phase: 0.2% w/v solution in chloroform Thickness : 0.5 mm
100.0 .
f---~_
80 --···-~I··--·_· __ ·_ · ·_·-t---
j
; ¡
60 J.___ .. ....._.--i-...............- - - J
40 ,~''',-,--_. -~~,~~.
i
~
Q)
o 20 i
e
ro I
::=
·E
,i
(J)
e
~
1- 0.0 t ._-~
¡
- -~~ .._-1-
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Diphenhydramine Hydrochloride RS450 Instrument: Fourier Transform Phase : Potassium Chloride Disc
100.0 '--~~ ..... _--_ . T - - --·
80
60
40
~ 20
e
ro
::=
·E
(J)
e
~
1- 0.0 ._- -+--
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Diphenylpyraline Hydrochloride RSI06 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0
80
60
40
~
e
20 --- ..~
:§
·E
(J)
e
~
1- 0.0 ____-i.-
2000.0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm-')
Dipipanone RSI07 Instrument: Dispersive Phase: Thin Film

100.0
80
60
40 4 _ _ _. __......_
<ft.
ID
u 20
e
ro
::='
-E
(J')
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Dipivefrine Hydrochloride RS382 Instrument: Fourier Transfonm Phase: Potassium Chloride Disc
100.0
40 ....._______---++ +
<ft.
ID
u 20
e
ro
::='
'E
(J')
e
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Dipyridamole RSI08 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60 ". .. ...... .......

" " ~
I
~._- ----_.
I
~ 20
e
:§
'E
(J')
e
~ 0.0 ----+---- ,,---"-
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V -S46 Infrared Referen ce Spectra 2014
Disod ium Pami dronate RSI09 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ,
80
60 i
40 + _______
;€
25 20 l
e
ro
::::
·E
<J)
e
ro
¡.=: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Disopyram ide RSIlO Instrument: Dispersive Phase : 10% w /v Solulion in Chlorofrm Thickness: 0.1mm
100.0 ,
¡
80
60
40 _I
<f!.
al
ü 20 .... ~.
e l
ro
::::
·E
<J) 1
e
ro
¡.=: 0.0 + I t
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Disulfi ram RSI1 1 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60
40 .
25 20
e
ro
::E
E
<J)
e
ro
¡.=: 0.0 ._ .. - ,. .. _ - -
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2014 Infrared Reference Spectra V -S4 7
Docusate Sodium RS1I2 Inslrument: Dispersive Phase: 10% wlv Solulion in Dichloromelhane Thickness : 0.1mm
100.0 T
I
80
60 __
40 .; -.- .- _.--i
~ 20
e
:§
·E
en
~ 0.0 1
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Domiphen Bromide RS383 Inslrumenl: Fourier Transform Phase: Polassium Bromide Disc
100.0
80
60
40
o~
Q)
ü 20
e
ro
-~
E
en
e
~ 0.0 ,." , i- .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Dopamine Hydrochloride RS 113 Inslrument: Dispersive Phase: Polassium Chloride Disc

100.0
80
60 ¡ ..1.._ .
I
I
40 ----- .~_ .. ~._- ~,-,.~ .. _.,
<ft. í
Q)
ü 20 ... 1
e
ro
::=
·E
en
e
~ 0.0 •. ---;-
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Dosulepin Hydrochloride RS1l4 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 T ........................
60 +-_..... ____ _
40
~
Q)
u 20
e
ro
~
E
(f)
e
ro
.= 0.0 ............--t-..
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Doxapram Hydrochloride RS116 Instrument: Dispersive Phase: Potassium Chloride Disc
?f2.
Q)
u 20
e
ro
:::::
'E
(f)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Doxepin Hydrochloride RS117 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
60
40
'1
~
o
Q)
u 20
e
ro
:::::
'E
(f)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Droperidol RS384 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
?f!.
al
u 20
e
ro
+='
'E
(J')
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Dydrogesterone RS118 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 + ............ .
40
?f!.
u
al 20
e
ro
+='
'E
(J')
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Edrophonium Chloride RS120 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
60
C
8 20 ¡
e
................... .
ro
+='
'E
(J')
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Ephedrine RS121 Instrument: Dispersive Phase: Thin Film

100.0
80
60
40
;ft
Q)
ü 20
e
:§
.E'
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ephedrine Hydrochloride RS436 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
80
60
o~
Q)
ü 20
e
ro
:::;
.E'
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Epinephrine (Adrenaline) RS122 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
80
60
40
~ 20
e
ro
:::;
.E'
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
2014 Infrared Reference Spectra V-SS1
Erythrornycin RS 123 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
40 { ............
<ft.
Q)
ü 20
c::
ro
:::::
'E
(f)
c::
ro
..= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Erythrornycin Estolate RS124 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80 +. ................ ........ ',
60 +,· .. ,···· ···· ···· · ··~·"~·t·- 4 ····· · ··· ..
40
<ft.
Q)
ü 20
c::
:§
'E
(f)
c::
ro
..= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Erythrornycin Ethyl Succinate RS125 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
80
60
40
~ 20
c::
ro
~
(f)
c::
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
V-S5 2 Infrared Reference Spectra 201 4
Erythrornycin Lactobionate RSl26 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
I
j
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Erythrornyci n Stearate RS127 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80 +-_._ .. ___ _
40
;f!.
Q)
u 20
e
ro
::=
'E
rJ)
e
~ I
f- 0.0 +
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Estrarnustine Sodiurn Phosphate RS 128 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0 ,_
40 J
I
8e 20
ro
::=
E
rJ)
e
~ 0.0 1. .. ___ -+ .....
2000.0 1800 1600 1200 1000 800 600 400.0
Wavenumber (cm-')
Estropipate RS129 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100,0
80
60
40
~
(!)
20
e'-'
ro
:::::
'E
(f)
e
ro
t= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Etacrynic Acid RS 130 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0
80
60
40
o~
(!)
20
e'-'
ro
-~
E
(f)
e
[:'
1- 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Etarniphylline RS 131 Instrument: Dispersive Phase: Liquid Paraffin Mull

100,0
80
60
40
25 20
e
fl
'E
en
e
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Etamiphylline Camsilate RSI32 Instrumenl: Dispersive Phase: Liquid Paraffin Mull

100.0 .
80
60
40
~
Cll
u 20
c
-~
E
(f)
c
II
ro
¡::: 0.0 i
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Ethambutol Hydrochloride RSI34 Instrumenl: Dispersive Phase: Potassium Chloride Disc

100.0
80
60
40
<f!.
Cll
u 20
c
ro
::=
·E
(f)
c
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ethyl Cinnamate RS136 Instrumenl: Dispersive Phase : Thin Film

100.0
80
60 i
r
40
~ 20
c
:§
·E
(f)
c
ro
.:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Etid.-onate Disodium RS440 Instrument: Fourier Transfonm Phase: Potassium Bromide Disc
100.0
80
60
40
~ 20
e
ro
:::;
'E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Etodolac RS 139 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
;f!.
Q)
u 20 i
e
ro
::=
'E
en
e
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Etoposide RS396 Instrument: Fourier Transfonm Phase : Potassium Bromide Disc

100.0
80 t--,.
60
40 ,
i
~ 20
e
ro
::=
'E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Etynodiol Diacetate RS138 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0 "1"'~~---'''~'''''-'~"~~''''-'''''-''~-''''''''''-''''''''''~'-r ..... ...- _ ..• ~-~_.~....
40 j- . ... ...........................¡:. _ \
<f'
(!)
t) 20
e
ro
:::::
'E
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Felbinac RS372 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100.0 T·······..·········•·········•····· ··:c ····· ··· ····································,.,. ............... ... .........................., .. ... ................. ... ........ .......... ....... -;-...•.
<f'
(!)
t) 20
e
ro
:::::
'E
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fenbufen RS140 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0 .,..······..··············_·_...··_·-T---··_.....·..·..···· ..····..·-r-·,...·.·····...__ ...__ .........r .............._-.~""_.,
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
2014 Infrared Reference Spectra V-SS7
Fenop.-ofen RSl41 Instrument: Dispersive Phase: Thin Film

100.0 , .
80
60
8e 20
ro
:::::
'E
(f)
e
~
f- 0.0 t---, ____ .'_._.______ ....__ .._j.... _ ... _•.•__ _
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Fe noprofen Calcium RSl42 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ,
I,
80
60
40
,,
j-
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Fcxofenadine Hydrochloride RS459 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
80
60
40
8e 20
:§
'E
(f)
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·1)
Flavoxate Hydrochloride RS143 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100,0 T" ., ' , .,.""."."", "",.".,,,.,,",,.,',.. . • , , . ' " " ..., . ",.. ············"''''T'··''···,' .... ,,, .... ',.", ,.".". , .,...... .. , .... " .... . T"" "
~
Q)
ü 20
e
ro
:::::
'E Cf)
e
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Flecainide Acetate RS397 Instrument: Fourier Transform Phase: Potassium Bromide Disc
;;F.
ü
Q)
20
e
ro
:::::
'E
Cf)
e
~ 0,0 ---..----t------ ...... o ••••••• _ •• _ . " •
,
.,¡
2000,0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm")
Flucloxacillin Magnesium RS144 Instrument: Dispersive Phase: Liquid Paraffin Mu ll

100,0 T ' '''''''''-'''''''''''''''''''''''1--' ''-''''''''''''-''''''''','' .... _.,""'. "'''''''''''''''''''',''- ' ' ' ' ' ' ,."''''',,.''' .....,,.T'''''-,... , . ... .. .
60 ...¡' '''''' ." ""-''''''''' 'f ''' """,;;rt''''''--,· '''+ , ,,,,,·_,,,,,·,, ,,,,,,··,,,t,,,,,·,,,,,,· .. ,, .., - , 11,"+"'''''' ' ''''''1'' 1'' 1
~ 20
e
ro
:::::
'E
Cf)
e
~ 0.0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm"')
Flucloxacillin Sodium RS145 Instrument: Dispersive Phase : Liquid Paraffin Mull
~
Q)
'-' 20
e
ro
:::::
'ECf)
e
ro
¡.=: 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-1)
Flucytosine RS146 Instrument: Dispersive Phase: Potassium Bromide Disc
~
Q)
'-' 20
e
ro
:::::
'E
Cf)
e
ro
¡.=: 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Fluocinolone Acetonide Dihydrate RS147 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
80 F=';':;;":;¡:=::::::~~" '"
40
~ 20
e
:§
'E
Cf)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-1)
Fluocinonide RS148 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
"#. ¡
~ 20 )
e
ro
:::::
·E
en
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
FluocOIiolone Hexanoate RS149 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .
80 +
i
60 +. __ .... __....~.. ... .
40 --¡
:
o~
Q)
u 20
e
ro
:::::
E
en
e ,
ro I
~ O.O ! ···r··
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fluorescein Sodium RSISI Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
60 .
i
40
~ 20 ¡
ro
:::::
E
en
e
~ 0.0 í
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fluorometholone RS152 Instrument: Fourier Transform Phase: Potassium Bromide Disc
<fi.
<l>
o 20
e
ro
~
'E
en
e
ro
r'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fluorouracil RS153 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T. '''''~''-'--'"'---''''
80
60
o~
<l>
o 20
e
:§
'E
en
e
ro
r'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fluoxetine RS398 Instrument: Fourier Transform Phase: Thin Film
60
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Fluoxetine Hydrochloride RS385 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100,0 T ..................................... ,
80
60
cf?
Q)
u 20
e
ro
::=
'E
(f)
e
~
r- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Flupentixol Decanoate RS154 Instrument: Dispersive Phase: Thin Film

100,0 ,......._ ................._..
80
60
40
cf?
Q)
u 20
e
ro
::=
'E
(f)
e
ro
¡.':: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Flu razepam Monohydrochloride RS155 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0,1mm
100,0
60
¡g 20
e
ro
::=
'E
(f)
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 ,0
Wavenumber (cm-')
20 14 Infrared Reference Spectra V-S 63
Flurbiprofen RS156 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
80
60
;1!.
Q)
u 20 ¡,
c::
:§
'E
(J)
c::
ro
r= 0.0 ,.
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm~l )
Flu rbiprofen Sodium RS157 Instrument: Dispersive Phase: Potassium Bromide Disc
100.0
80
60
I
40 .1.
;1!.
8c:: 20 1
ro
:::e
'E
(J)
c::
ro
r= 0.0 i
.+
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm~l )
Fluticasone Propionate RS158 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0 _
80 .;
60 l'
8 20 .... .• -t .
c::
ro
:::e
'E
(J)
c::
r=ro 0.0 .¡_ " ____ ' ___ _ . . . . . . . ~-- {-_o
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm~l )
Fluvoxamine Maleate RS159 Inslrument: Fourier Transform Phase: Polassium Bromide Disc
<f<.
Cll
o 20
e
ro
::::<
'E
en
e
ro
t'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Foscarnet Sodium RS 160 Inslrument: Fourier Transform Phase: Polassium Bromide Disc
<f<.
Cll
o 20
e
ro
::::<
'E
en
e
~
f-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Fosfestrol Sodium RS161 Inslrument: Dispersive Phase: Liquid Paraffin Mull
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1
)
Fumaric Acid RS163 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
;f?
Q)
u 20
e
ro
::=
"E
(j)
e
~
t- 0.0 1
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Furazolidone RS164 Instrument: Dispersive Phase: Pptassium Bromide Disc

100.0 ,
80
40
;f?
Q)
u
e
:§
"E
(j)
e
ro
¡.=: O.Oi
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Fusidic Acid RS 166 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0.1mm
100.0 . "f
80 .j___ "
I
60 !
I
40 ~_ _. __ . ___ ,___
;f?
Q)
u 20
e
:§
"E
(j)
e
,
I
~ 0.0 ' L
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
V-S 66 Infrared Reference Spectra 201 4
Gernfi brozil RS167 Instrumen\: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 ¡
40
¡
o~ I
Q)
'-'
e
20 J-- I
ro
:::::
'E
rJ)
e
ro
.= 0.0 ¡_ .. ,
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Gliclazide RS168 Instrumen\: Dispersive Phase: Potassium Bromide Disc

100.0
60 i I
40
~ I
Q)
'-' 20 1-,
e
ro
::::: ,i
'E
rJ)
e
ro
.= o.oJ __.__
I
-¡--
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Glirnepi ride RS463 Instrumen\: Fourier Transform Phase: Potassium Bromide Disc
100.0 .. __..~ ...__ . __..._..<_~._.. ____ _
80
60
,1
8 20
e
ro
:::::
'E
rJ)
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Glipizide RS169 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0 ...... ..._
¡
80 J _.~._
60
40
cf!. '
2l 20
c:
L.-- .
I
.~
E
en
c:
ro
¡:: 0.0 + .. ~_..... .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
Gliquidone RS170 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100.0
80
40
i
I
O.O.! ...... _ _... -t· - .-.-..... _--:
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm·')
Glycine RS171 Inslrument: Dispersive Phase: POlassium Bromide Disc

100.0
80
60 '
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm·')
Griseofulvin RSl72 Instrument: Fourier Transform Phase: 1.5% wlv Solution in Chloroform Thickness: 0.1mm
100.0
80 +_ ... .•.... ~._......
60
40
<F.
Q)
u 20
e
g
"E
m
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Haloperidol RS173 Instrument: Dispersive Phase: Potassium Chloride Disc
100.0 . ...... . . . . ...... ...
-w• • • • • • • ~._. .. . . ._ . " T -.. ..•.
<F.
Q)
u 20
e
ro
:::;
'E
m
e
ro
¡::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Hexachlorophene RS174 Instrument: Dispersive Phase : Potassium Bromide Disc
100.0
80
60
40
~ 20
e
ro
:::;
"E
m
e
ro
¡::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Homatropine RS175 Instrumenl: Dispersive Phase: Thin Film

100.0
60 t .......................................... ¡
40
,
I
~ 20 _!__ _
e:
g
·E
en
e:
.=ro
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Hydralazine RS176 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0 r ..
,
,
60 +___ _
i
40
~ 20
e:
~
E
en
e:
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Hydralazine Hydrochloride RSl77 Instrumenl: Dispersive Phase: Potassium Chloride Disc

100,0
80
60 ..¡...........................
0.0 - ¡ - -
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Hydroch lorothiazide RS178 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0 __ ,
80
60
¡
40 -l., _ . _. -~-'- -'-' ._--"...- -, --
!
u
Q) 20 ,i
e
ro
:::::
'E
en
e
.=ro 0,0 ~, .. ~--- ,
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm- 1
)
Hydrocortisone Acetate RS179 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0 y _ """'" _,
80
60
~
Q)
u 20
e
ro
:::::
'E
en
e
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-1)
Hydrocortisone Sodi um Phosphate RS386 Instrument: Fourier Transform Phase : Potassium Bromide Disc
100,0 ,_
60
8e 20
ro
:::::
E
en
e
.=ro 0,0 ,;__,_____ ..
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-1)
Hydrocortisone Sodium Succinate RS180 [nstrumen!: Dispersive Phase: Potassium Bromide Disc
1000
~
Q)
ü 20
c::
ro
:::e
'E
en
c::
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Hydroflumethiazide RS181 [nstrumen!: Dispersive Phase : Potassium Bromide Disc

100.0
?f2.
Q)
ü 20
c::
ro
E
E
en
c::
.=ro 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm- 1
)
Hydroxycarbamide RS184 [nstrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
¡:s 20
c::
ro
:::e
'E
en
c::
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1
)
Hydroxychloroquine RS182 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1mm
100.0 r ................ " .. " ........."""""" ..,
60
'cF.
(j)
o 20
e
ro
::=
'E
rJ)
c:
~
r- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Hyoscine Butylbromide RS185 Instrument: Dispersive Phase: Potassium Bromide Disc

100. O T" ...." ....".... """"1"""" "" " " " """"""""""""","""""" """""""""
40 +....._ """"""""" "
'cF.
(j)
o 20
e
ro
::=
'E
rJ)
c:
~ 0,,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ibuprofen RS186 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60
~ 20
c:
ro
::=
"E
rJ)
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Ifosfamide RS428 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
60
40 + ."". """",, ,,'
<ft.
(J)
'-' 20
e
:§
'E
en
e
ro
r= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Indometacin RS187 Instrument: Dispersive Phase: Liquid Paraftin Mull

100.0
80
60
40
>!i!.
o
(J)
u 20
e
:§
'E
en
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Indoramin RS188 Instrument: Fourier Transform Phase : Potassium Bromide Disc

100.0
80
60
40
~ 20
e
ro
:::::
'E
en
e
~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Inositol Nicotinate RS190 Inslrument: Dispersive Phase: Polassium Bromide Disc
60
?ft
CIl
o 20
e
~
E
(f)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
lodipamide RS191 Inslrument: Dispersive Phase: Liquid Paraffin Mull

100.0
80
60
40
~
CIl
O 20
e
ro
:::::
·E
(f)
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
lopamidol RS441 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100.0
60
1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
lopanoic Acid RSl92 Instrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
;f<.
Q)
o 20
e
:§
'E
en
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400 .0
Wavenumber (cm- 1
)
Irbesartan RS467 Instrumen!: Fourier Transform Phase: Potassium Bromide Disc
80
60
40
;f<.
o
Q)
20
e
ro
.E
E
en
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Isometheptene RS195 Instrumen!: Dispersive Phase: Thin Film
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Isoniazid RS196 Instrument: Dispersive Phase: Potassium Bromide Disc
<f!.
Q)
u 20
e
g
·E
rJ)
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Isosorbide Dinitrate RS412 Instrument: Dispersive Phase: Potassium Bromide Disc
80
60
40
2l 20
e
ro
:::::
E
rJ)
e
ro
.= 0.0 ._.~-------+-_ ... _-- -.. __.__ ._-;-.
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
lsradipine RS373 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
60
2l 20
e
ro
::::<
-E
rJ)
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ketoprofen RS198 Instrument: Dispersive Phase: Potassium Bromide Disc
o~
al
() 20
e
ro
::=
'E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Labetalol RS199 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 ¡ ................ ..... . ..
40
~
al
() 20
e
ro
::=
'E
en
e
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Lacidipine RS407 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

100.0
60
§ 20 . . . ........ ... ...... .

ro
::=
'E
en
e
~ O.O + ..~~•.
2000.0 1800 1600 1400 1200 1000 800 600 400 .0
Wavenumber (cm-')
Lamivud ine RS451 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
<f2.
ID
o 20
e
ro
::::::
'E
en
e
ro
¡.':::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Levobunolol Hydrochloride RS200 Instrument: Dispersive Phase: Liquid Paraffin Mull
<f2.
ID
o 20
e
ro
::::::
'E
en
e
ro
¡.':::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Levodopa RS201 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
2l 20
e
ro
::::::
'E
en
e
ro
¡.':::: 0.0 +_ ... ~.~~ __ ........ .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Levomepromazine RS404 Instrument: Fourier Transform Phase : Liquid Paraffin Mull

1000
80
60
40
?f!.
Q)
u 20
c:
g
'E (f)
c:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Lidocaine (1) RS202 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ".. " ,~ . ~, ..
60 . . ... . .... .
40
?f!.
Q)
u 20
e
g
'E
(f)
e
ro
~ 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Lidocaine (2) RS405 Instrument: Fourier Transferm Phase: Film en Potassium Bromide Disc
100.0
80
60
40
~ 20
e
g
'E
(f)
e
~ 0,0
2000 ,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Lincomycin Hydrochloride RS203 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 .,.............................
80
60
<f!.
Q)
u 20
e
ro
::=
·E
(f)
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Lofepramine Hydrochloride (Form A) RS399A Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
60
40
<f!.
Q)
u 20
e
ro
::=
·E
(f)
e
ro
.=
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Lofepramine Hydrochloride (Form B) RS399B Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
60 . j ............•...•..•........••_ .... + .____ .. ~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
20 14 Infrared Reference Spectra V -S8 1
Lom ustine RS204 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ·c · _............... • ...... ......... _ - ._-..... .. 'í ......._. . . .
I
80
60
40 i
I
• ~_'u~ ou¡.. •
I
._-¡
II
cF-
Q)
ü 20
I
~
e
ro
:::::
'E
en
e
ro
.= 0.0 + ...•.. ..
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Loprazolam Mesilate RS20S Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 -r.. ... ........ . ........_.. _ ....... r . . . . . . . . . . . . . . . , ..
¡
I
80 ~ ...._ .... _...._.
60
40
cF-
Q)
ü 20
e
:§
'E
en
e
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Loratad ine RS43S Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ....
[.
I
40 .:.....__.._ .... _ _._.......
~
~ 20
e
ro
:::::
'E
en
e
~ 0.0 ... _-Í-
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Lorazepam RS206 Instrument Dispersive Phase: Potassium Bromide Disc
~
o
<D
ü 20
e
ro
:::::
'E
(f)
e
ro
!-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Lormetazepam RS207 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1
)
Losartan RS453 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
2000.0 1800 1600 1400 1200 1000 600 400.0

Wavenumber (cm- 1
)
Mebeverine RS208 Instrument: Dispersive Phase: Thin Film
<f!.
Q.)
u 20
e
ro
:l::'
·E
(J)
e
ro
¡:: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mebeverine Hydrochloride RS209 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
I
80
60
40
<f!.
Q.)
u 20
e
:§
·E
(J)
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Medroxyprogesterone Acetate RS421 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
80
60
40
25 20
e
:§
·E
(J)
e
ro
..::: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
100.0
Mefenamic Acid RS210
. . . . . . . _- Instrument: Dispersive
.... r .
Phase: Liquid Paraffin Mull
r l
80
60
40 -r-----+----
<f!.
ID 20
u
e
ro
:::::
'E(j)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Megestrol Acetate RS211 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
80
60 +..-.. ----.......---.--......-~¡- _t
40 t.-
I
<f!.
ID 20
u
e
ro
:::::
'E
(j)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Melatonin RS455 Instrument: Fourier Transform Phase: Potassium Bromide Disc
60 l' "-.-.-..
!
I
40 -t-.......-----+----...... ,,---.-...-..M------;·-·-- ·--·--·----l -y---'---'-.-.•. -'- _.~--- _._ .....----- -.---__.... .
I
0.0 _j._ .. __ .. _.. ¡-
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Meloxicam RS374 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 ,
80 +.,........ _._ ...........
60
40 .~'_.,.
cf!.
§ 20 .}
~
E
rf)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Melphalan RS212 Instnument: Fourier Transform Phase: Potassium Bromide Disc

100.0 .
80
60 _¡-
,
40 1.
I I
8 20 ¡
e
ro
:::::
'E
rf)
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Menadiol Sodium Phosphate RS213 Instnument: Dispersive Phase: Potassium Bromide Disc
100.0
60
40 .,
I
I
0.0 l._ .+------_.
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Menadione RS214 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
cf?
Q)
() 20
c:
ro
~
E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Meptazinol Hydrochloride RS21S Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
80
60
~
Q)
() 20
c:
ro
::::
E
en
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mepyramine Maleate RS216 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0
60
40
0.0
I
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- t )
20 14 Infrared Reference Spectra V-SS?
Mercaptopurine RS426 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 ....
80 t
------1
60
40
25 20
c::
ro
~
'E
(j)
c::
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Mesalazine RS454 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
60
40
<f?
ID
u 20
c::
ro
~
'E
(j)
c::
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 600 400.0
Wavenumber (cm- 1
)
Metformin Hydmchl oride RS217 Instrument: Dispersive Phase: Potassium Chloride Disc
100.0
80
60
40
25 20
c::
ro
~
'E
(j)
c:
~ 0.0 J
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Methadone RS218 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ..,--_________,,--_. ______ ....,_.
40
~
e
<1>
(,) 20 ._ -_ ....... . ._- -J f
ro
::::<
'E
en
e
~
t- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Methanol RS387 Instrument: Fourier Transform Phase: Thin Film

100.0
80
60 r . · . · -·..·. ·····........··..·f \- ........
40
o~
<1>
(,) 20
e
ro
~en
e
~ 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-')
~
e
20 +___. _._.__-+___..._______~_----~---~.-----.----j----,,- .... .--1 . --
~
E
e'"
ro
.= 0.0 +--------+ ...------t--.------t----------+-.-.. . _. . ____ .+-_.. . - - - _ t----- _. -
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Methoxamine Hydrochloride RS220 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 ,.. .................................
80
60
40
e!
B 20
e
ro
::::<
"E
en
e
ro
.= 0,0 .1...... ··w··_··, ... ·· '~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Methyl Nicotinate RS222 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0
80
60
40
B
e
20
:§
"E
en
e
ro
¡.':: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Methyldopa RS223 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .. "
,
80
60
'#- ¡
§ 20 t-
1,,1 2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Methyldopate Hydrochloride RS224 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
~ 20 +--..--...-.-.~r-·--lf----·-·~-- 4~-----·~---·-·---····..·--"~·.-.-.--.----..+--......- .-...--. ,

e
g
'E
en
e
(1)
.= 0.0 +...__.._..__.._. . + ........................... -+..................................- t .......... --- .............+ .._ .........---.....+ ... .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Methylphenobarbital RS388 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.OT .. ·· .... ·· ........ ···· ..........·.. ·· .... ·:: .......... ··.. · ....·· .. ··•· ..· ..···']"·...... ·· .. ·...... ··..·.................. · .. ,..·............ ·· ........·.... •·· ..· .. ·.. r ................
60 .¡---......----.-.J.-+......
o~
Q)
o 20
c::
(1)
:::::
'E
en
c::
(1)
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Methylprednisolone RS225 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 . ,......................................-., .........--..........-.... r ..........._ ..................- , ..- -....- ...- ...........- ....., ..............._ ..................................-..._ ...... .
~
~
c::
20 7· ....·······....·.. ·....·.. ·;····_· ...·_ ...... ,·' .....tl--......···........---.+ ..--.~-....-~........__......-._.-_. .r_ ...... - -
g
'E
en
c::
~ 0.0 + ............ ..........._-....f-._ ...............- ...+ ... - .... ____ ..__._,. _...........__ ._... _.+.......___ .... __ ..
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
2014 Infrared Reference Spectra V-S9 1
Methylprednisolone Acetate RS226 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .-_._____,...-_ _ ._~_-.-- . ¡
80
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Methysergide Maleate RS227 Instrument: Dispersive Phase: Potassium Brom ide Disc
100.0 T--- -r
I !
I ¡
80 i
I
60 ~
I
40 .~. .
;¡¿
Cll
o 20
e
ro
::::
'E
<n
e
ro
¡':: 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Metipranolol RS375 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 "T
I
I
60
40 +_______+__ ._ . .• ' . ___ +I

j
25 20
e
ro
::::
'E
<n
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Metoprolol RS228 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0 . . , . ...
80
60
40
cf!.
Q)
Ü 20 .~t-.,.
e
ro
::::<
·E
(f)
e
ro
.= 0.0 . i·- ••.• _ . u_ . _ _ ..•.. -'_
.~ . .- .. ~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Metronidazole RS229 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
cf!.
Q)
ü 20
e
ro
::::<
·E
(f)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Metronidazole Benzoate RS408 Inslrument: Fourier Transform Phase : Polassium Bromide Disc
100.0 ...
¡ r
,!
I
60 ·····l
40
I
I
0.0 <
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Metyrapone (1) RS230 Instrumenl: Dispersive Phase: Liquid Paraffin Mull

100.0 . j ....."" ........................................" ...... , ........ ".......... ".. " ................ ".. _ ............, .... "............. ..
40 "~. _ _-----~----+-+-+r"r---
;g
o
Q)
'-' 20
e
ro
::::::
"E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mety rapone (2) RS231 Instrumenl: Dispersive Phase: 50% w/v Solution in Macrogol 400 Thickness : O"1mm
100.0 . ,. . . ." . . . . """ . . . . . . . . . . . . . . . . . . . . . ". :. . . . . . , . . .,,_" """ . """ ........ ", . " . . "" . . . . . "" ........................". ","
?fl..
Q)
'-' 20
e
:§
"E
en
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mexenone RS232 Instrumenl: Dispersive Phase : Potassium Bromide Disc

100.0
T
60 +1 __ . .___ _
40
1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Mexiletine Hydrochloride RS233 Instrument: Dispersive Phase: Potassium Chloride Disc

100,0
1
80 t - -
I
60 f
u
Q)
20
e
:§
'E
(fJ
e
ro
.= 0,0 1. .,............_+-_.., ,
+ ·-·-t
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm")
Mianserin Hydrochloride RS234 Instrument: Dispersive Phase: Potassium Chloride Disc
2000,0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm")
Midazolam RS235 Instrument: Dispersive Phase: Potassium Bromide Disc
~ 20
e
ro
:::::
'E
(fJ
e
ro
.= O,O ~. _
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm")
Minoxidil RS376 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 _,
r
80
60
_. - 'o _ _ _""'j"' H _ _ _ _ _ o_ _ _ _ o_ _ _
;f!.
Q)
ü 20 --- -1
e
ro I
:::e
°E
en
e
~
1- 0.0 ..,__ '1'
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
r --
Mirtazapine RS434 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ........ · ..··1
-¡ --
60
8e 20
ro
:::e
°E
en
e
ro
¡.':: O.o .t·
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Mitobronitol RS236 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 ,
60
1
40 "",0 "O
gro 20 +
:::e
°E
(f)
e
1, !
ro
.= 0.0 +-__ . y _ _ •• y _
J • _ _ _ _ .0' __
I
-j-o_
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm" )
Morphine RS237 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60
o~
al
u 20
c:
ro
:::::
'E
en
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Moxisylyte Hydroehloride RS238 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
60
<f!.
al
u 20
c:
ro
:::::
'E
en
c:
~
1- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mucie Acid RS377 Instrument: Fourier Transform Phase: Potassium Bromide Disc
60 f" ""'"'''''' '''''''''''''''''''' ''''
25 20
c:
ro
:::::
'E
en
c:
~ O.O +~_________~~ _____ ..~ ___~___ "__"__._.__4-"._.. _.._"__~__________~________-+.__________~'"._______.._~
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Mupirocin RS425 Instrument: Fourier Transform Phase: thin film

100.0
80
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Nabumetone RS239 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80 {
60
I
I
40 ~ "+¡
~
<ll
o 20
e
ro
~
E
CFl
e
.=ro 0.0 L.. , 1_ "
2000,0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm"')
Naftidrofuryl RS240 Instrument: Fouri er Transform Phase : Thin Film

100.0
80
60
~ 20
e
ro
:::::
'E
CFl
e
.=ro 0,0 :. ______ _ -1
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Nalidixic Acid RS241 Instrument: Dispersive Phase: Potassium Bromide Disc

100,0
80
60
40
~ 20
c:
ro
:::::
'E
Cf)
e
ro
~ 0.0 - ¡ - - - , - - - _..-+-,...
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm")
Nandrolone Decanoate RS242 Instrument: FTIR Phase: 10%w/v Solution in Dichloromethane Thickness: 0,1 mm
100,0 ,_ __ o
80
40
~ 20
c:
ro
:::::
'E
Cf)
c:
ro
~ 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-')
100, O """"'--'-'-'=~ .- .. ¡ ..__ ._-~
' ~ ..,
V
rv', !
i
'
80 J '
60
I
L J '
40
~ 20
e
ro
:::::
'E
Cf)
c:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Nandrolone Phenylpropionate RS243 Inslrument: FTIR Phase: 10%w/v Solulion in Dichloromelhane Thickness : O.1mm
100.0
80
60
40
?f2.
u
Q)
20
c:
ro
:t::'
'E
(J)
c:
ro
.= 0.0
4000.0 3600 3200 2800 2400 2000.0
Wavenumber (cm-!)
100.0 _, __ o • _ _ _. _ • • • • _ _•••_ •• _ ••• _ • • _ _ _ _• • _~---- . .- - . - - - . ; . - . ' _ . _ __ ~_, _ _ _ '_._
80
60 ·· _·__· · _-t-·
40 ........ ... ..
~
I
.J . ... . . .. . .. . . . . . . .. .(
~ 20 ... __
c:
ro
:t::'
'E
(J)
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-!)
Naproxen RS244 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
r _··- _.
60
.--------" ~~t··
I
?f2.
Q)
u 20
c:
ro
:t::'
'E
(J)
c:
~ 0.0 -+
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-!)
Niclosamide RS245 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
<f!.
Q)
ü 20 .j
e
ro
::::
·E
rJ)
e
ro
t= 0.0 !
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Nicorandil RS461 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
60
40 .... L.
~ 20
e
ro
:¡:::
·E
rJ)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm· 1)
Nicotinamide RS246 Instnument: Dispersive Phase: Potassium Bromide Disc

100.0
T
I
I
80
60
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-1)
2014 Infrared Reference Spectra V-S 1O1
Nicotine RS452 Instrument: Fourier Transform Phase: 0.5% w/v Solution in Chloroform
<f2.
Q)
ü 20
e
ro
::=:
'E
(J)
e
¡::
1- 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Nifedipine RS248 Instrument: Dispersive Phase: Potassium Bromide Disc
<f2.
Q)
ü 20
e
ro
::=:
'E
(J)
e
ro
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Nikethamide RS249 Instrument: Dispersive Phase: Thin Film

100.0
60 + ................ . ... . . .. .
40
~ 20
e
g
'E
(J)
e
~ O.O+ ....~_.
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Nizatidine RS410 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
60 ,
2l 20
e
ro
~
"E (f)
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ondansetron RS418 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 . T--
I
40 t
¡
<F-
2l 20
I
j
1
.:: 0.0 '1~""
2000.0
I
1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Ondansetron Hydrochloride RS427 Instrument: Fourier Transform Phase : Potassium Chloride Disc
100.0
80 _ •• '" ~",.H" •• ~. __ ~
T----
I
I
60 t
40 ·'·W'_ _ ~Y_· .·_,·,
" '~"~-'-~ --
<F-
Q)
ü 20
e
ro
~
·E
(f)
e
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Oxazepam RS253 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
80 ..
60
40 ..: _ ..
~ 20
c:
ro
E
E
en
c:
ro
.= 0.0 ..i- ._._•.......... _.. _._... _.--....r ••.•- -..•••••.•
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Oxetacaine RS254 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0
80
60
,
40 I
+ ..-
i
~
Q)
o 20 .
c:
ro
::=
'E
en
c:
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Oxprenolol RS255 Inslrument: Dispersive Phase : Polassium Bromide Disc

100.0
80
60
~ 20 .·
c:
ro
::=
'E
en
c:
ro
.= 0.0 :
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Oxybutynin RS442 Instrument: Fourier Transform Phase: Thin Film

100,0
o~
al
ü 20
c::
ro
:l='
'E
en
c::
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Oxycodone RS457 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100,0 '} .............. . . . . . . . . .
60
40
~ 20
e
:§
'E
en
e
~
1- 0,0 ...... "-r ...
2000,0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm- 1)
Oxymetholone RS256 Instrumen!: Dispersive Phase: Potassium Bromide Disc

100,0
80
60
40 +-_. ___...__ .___ . . _. ,j" ...
~
~ 20
e
ro
:l='
'E
en
e
ro
í
~ 0.0 - ~- -,1
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
2014 Infrared Reference Spectra V-S10S
Papaverine Hydrochloride RS415 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100,0
80
40
20 j " """"""""",,,,,,,,,,,, ,:
0.0
2000,0 1800 1600 1400 1200 1000 800 600 400.0
Paracetamol RS258 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~
(!)
ü 20
e
ro
::=
'E
(J)
e
~ 0,0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pentamidine Isetionate RS259 Instrument: Dispersive Phase: Liquid Paraffin Mull

100,0
60
40
1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm-')
v -S 106 Infrared Reference Spectra 2014
Pentazocine (Form B) RS261 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .r .......•................•..
60
-¿ft
Q)
u 20
e
ro
:l='
·E
en
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pentazoci ne Lactate RS263 Instrument: Dispersive Phase: Potassium Bromide Disc
o~
Q)
u 20
e
ro
:l='
·E
en
e
~ 0.0 .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pentobarbital RS264 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T
~
80
60 + ...................... ..... ... .+ \ .............. .. .., .. .. ~.
40
25 20
e
ro
:l='
'E
en
e
~
f- 0.0 ----¡----- --~
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Perphenazíne RS265 Instrument: Dispersive Phase: 5% w/v solution in Chloroform Thickness : 0.1mm
100.O~__ _____ ___.,-__ __
80
60 .
40 L
;¡¿
Cl)
o 20
e
ro
::=<
'E
en
e
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pethídíne RS266 Instrument: Dispersive Phase: Thin Film

100.0
80
60
I
I
40 -!i
~--
I
;¡¿
Cl)
o 20
e
ro
::=<
'E
en
e
ro
t= 0.0 . _-_..¡- - -,--~ ' - - .--.. .¡
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Pethídíne Hydrochloríde RS267 Instrument: Dispersive Phase : Potassium Chloride Disc

100.0
l
80
60
g 20 +___ .... _........

ro
::=<
'E
en
e
~ 0.0 ._ _ _ _ .. -..¡.----.---..--,---_.-.- -t---..
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Phenindione RS268 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40 +_ ...... _._ ..._ ..
~ 20
c:
ro
:::::
'E(f)
c:
~
1- 0.0 --_.. ;. - ~_ . _----_._+. _-_ . ._.. ---
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Phenobarbital RS270 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60
4O-t-.. -............. ---...
<f<.
Q)
'-' 20
c:
ro
:::::
'E
(f)
c:
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm")
Phenoxybenzamine Hydrochloride RS271 Instrument: Dispersive Phase: Potassium Chloride Disc

100. O-r----.--. .. -. ___ . ,...... _.........____ ... __._" . __....._ .._ .... , •......._._ .....•.._...•...,._......... ____. _....•"_ __ ._......___...___ ,__ _
60
1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm")
Phenytoin RS272 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T - ..-----.. - - .......... - .. .. - ............... - .. -.. ... .........}
,¡ ¡
60 . ¡ ............ ................ ........... . ++........... + ...... .+......-.. ... . . . .. ......... u . . ·. ¡,I · .. ·..·-···· , ... , . .. ': ... -
e:.
ü
Q)
20
e
g
'E
en
e
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pholcodine RS273 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60 1
., .............. ,.. ,...., . _ _ -+ . . .". . ......... . ················ · 11
<ft.
Q)
ü 20
e
ro
:::::
'E
en
e
~
1-- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Pilocarpine Nitrate RS274 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0 T .................................... , ....... , .....................
60 +, ................................ , \;.......
~ 20
e
g
'E
en
e
~ O.Oy-__ ._~. ____.~~______y __..___.. _____+-_________ ~ ___________ +. ______.__~ __
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
v-S 11 O Infrared Reference Spectra 2014
Pimozide RS389 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

100.0 ~. ___ _
I
I
80
60
40 +_ ___ ---1- __
I
~ 20
e
ro
::=
·E
rJ)
e
~
f- 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-') ,"
Pindolol RS275 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0 T
80
I
60
I
40 I J.
I
~
1
I
Q)
ü 20 ;
e
ro
::=
·E
I
rJ)
e
ro
I
~ O.OJ i
-t !
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pizotifen RS276 Inslrument: Dispersive Phase: Polassium Bromide Disc

100.0 .
80
60 .... L.
1
I
40 .._._- ~i~·
J..,....".. _ _ ~
-~ .. _~._~ .. . j
~
Q)
ü 20
e
ro
::=
·E
rJ)
1
e I
ro
~ o.oL __ --i
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pizotifen Malate RS277 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 T ························
i
--.1
i
40
r
<ft.
ID
ü 20
c:
ro
:::;
'E I
en
c:
ro
.= 0.0 J . ~ ...
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Poldine Metilsulfate RS278 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 ·············.. ··1
80 _¡
60
I
I
40 +-
1200 1000 800 600 400.0

Wavenumber (cm-')
Polythiazide RS280 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
40 ]._
I
<ft.
~
:t:
20 i.
'E
en
c:
ro
.= 0.0 .:...
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Prazosin RS281 Instrumenl: Dispersive Phase: Potassium Bromide Disc
60
40 L
!
<f2.
al
o 20
c::
:§
'E
<n
c::
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- t )
Prednisolone RS282 Instrumenl: Dispersive Phase: Potassium Bromide Disc
<f2.
o
al 20
c::
ro
+='
'E
<n
c::
~
t-- 0.0
2000.0 '\.800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-t )
Prilocaine RS285 Instrumenl: Dispersive Phase: Thin Film
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-t )
Primidone RS286 Inslrumen!: Dispersive Phase: Polassium Bromide Disc
~
(l)
o 20
e
ro
:l='
-E
en
e
ro
~ 0.0
2000_0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Probenecid RS287 Inslrumen!: Dispersive Phase: Polassium Bromide Disc

100.0 T---. -- ---
60
40
o
Cll 20
e
ro
:l='
'E
en
e
ro
~ o-o
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Procainamide RS288 Inslrumen!: Dispersive Phase : 5% wlv Solulion in Chloroform Thickness: 0_1mm
2000.0 1800 1600 1400 1200 1000 800 600 400 .0

Wavenumber (cm-')
Prochlorperazine (1) RS289 Instrument: Dispersive Phase: 10% w/v Solution in Chloroform Thickness: 0.1mm
100.0
80 -l---_...........-......_+_.................""" .". . .......:.
60
40 ¡... .. . "" .... ". "',,_'
I
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Prochlorperazine (2) RS390 Instrument: Fourier Transform Phase: Thin Film

100.0
80
g 20 +~~__~__~+_ ....__.. " "..,,___ . _ ....".....-r~...+.._. . . ". _-.... ""..". ""'_. . . . "...._. , ._- :"
¡g
'E
<n
e
ro
~ 0.0 ¡ ............. _............. +._.__ ........". __.... ) ...". "..._ ...... .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Prochlorperazine Mesilate RS290 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0 r -....-..."""--,,.............-, .. ,,
60
1800 1600 1400 1200 1000 800 600 400 . 0

Wavenumber (cm-')
Procyclidine RS291 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T~'~~~~~" '-"'-' ~.,. ___ ".,.~
60 ~~
40
~
(j)
ü 20
e
ro
::::::
'E
en
e
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Procyclidine Hydrochloride RS292 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
I
I
80
60
40
~
(j)
ü 20
e
:§
'E
en
e
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber ( cm~' )
Progesterone RS293 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
2l 20
e
ro
:E
E
en
e
ro
t= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Proguanil Hydrochloride RS294 Instrument: Dispersive Phase: Potassium Chloride Disc
;g
o
Q)
o 20
e
ro
:::::
°E
en
e
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm" )
Promazine RS295 Instrument: Dispersive Phase : 5% wlv Solution in Chloroform Thickness: 0.1 mm
100,0 T " " " ' - ' ,:: "'"'''''''''''' ....... ,"';--.. ,.. ......" .. , ' ...... " " ' ' Y ' ' ' ' ' ' ' ' ' '' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' '
""'" • "'.'.",,_.- ,,'o """.",,, ._ j...,,,

2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm")
Promethazine RS297 Instrument: Dispersive Phase: 5% wlv Solution in Ch loroform Thickness : 0. 1mm
100,0 ,
60
1800 1600
J
1400 1200 1000 800 600 400,0
Wavenumber (cm")
2014 Infrared Reference Spectra V-S 11 7
Propofol RS416 Instrument: Fourier Transfonrn Phase: Thin Film

100,0
60
20
o, O":""'""' '' '''''' ""' "~,o ..'"

2000,0 1800 1600 1400 1200 1000 800 600 400,0
Propylene Glycol RS437 Instrument: Fourier Transfonrn Phase: 40% wlv Solution in Water Thickness 0,5 mm
100,0 ,,'
80
60
40
~
Q)
ü 20
e
ro
~
'E
rJ)
e
ro
.= 0,0
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm")
Propranolol RS298 Instrument: Dispersive Phase: Potassium Bromide Disc
2000,0 1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm" )
Propylthiouracil RS300 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
?ft
Cll
o 20
c:
ro
:::::
·E
en
c:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Protriptyline RS301 Instrument: Dispersive Phase : Thin Film

100.0 T .._ .. ~--..- ..........-) ..... .....
80
60 +_____~. . ........... _
40
<f<
Cll
o 20
c:
ro
:::::
-E
en
c:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Proxymetacaine RS302 Instrument: Dispersive Phase: Th in Film

100.0
80
60
40
~ 20
c:
~
E
en
c:
ro
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Proxyrnetacaine Hydrochloridc RS303 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
60 .
40 ¡..
~
Q)
() 20 .,. r
e
ro
:t:'
-E
(J)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pseud oephedr-ine RS304 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Pyrazinarnidc (1) RS306 Instrument: Dispersive Phase : Potassium Bromide Disc
Q)
()
e
ro
:E
E
(J)
e
.=ro
Wavenumber (cm-')
Pyrazinamide (2) RS438 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
60 j----.--- .. --+ \ .-------------------.--------------- -t----- ------[
?f2.
Q)
o 20
e
:§
-E
en
e
ro
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pyridoxine Hydrochloride RS308 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 ---:----~---~----r__----~----------,--~----------~.-----,--- ---------- -..,----.------------------.------ - --~- - r--
60
~
Q)
o 20
e
ro
~
'E
en
e
ro
t-'= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Pyrimethamine RS309 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .,.----.-------------.-.--._--.------y-------____ -- . ------T.----- ---_._ .___________.__
~ 20 +_____________ . ____ __________ + ______ ______ ._.___ - tl!'+ l-. -jl - ---- j/ .i--
e
:§
'E
en
e
~
t-- 0.0 -+-----+--------+------+-----~_____1~------!----- _____¡ - - __ _ _ ~-
2000.0 1800 1600 1400 1200 1000 800 600 400_0
Wavenumber (cm-')
Quinolin-8-ol RS310 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0 , __ _
I
80
60
40
o~
<ll
ü 20
e
ro
:::::
'E
en
e
ro
.= 0.0 j
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Ramipril RS417 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0 __ . . ........................._
80 i-
60
20 + ..... _. . . __ ........__
0.0 ~_______.___ ,._.,,_--'_.____ "" . _._

2000.0 1800 1600 1400 1200 1000 800 600 400.0
Ranitidine Hydrochloride RS311 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0 r'"
80
60
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Ribavirin RS349 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

100, OT··"·······",,· '''''''''''''''''' ''' ''''''' , ' '''''' '' '' '''''''''''''''''''''''''' '' '1'' . " ' " ."" .• " ". " •• •• •• ,, " ' " . " ... ... " . " " ' " ' '''''''''' ' ''T'''''' '''''''' '' ' '''' '' ''''''''''''''' '' ''' "·c"""· ·
~
Q)
u 20
e
ro
:::::
'E
U)
e
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm"')
Rifampicin RS312 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100,0 'T"".''''''''.".-''-----,,''''''''.~-'--''~''''-''..,-
80
60 +--'-'-"-r-"-f
40
?fi.
Q)
u 20
e
g
'E
U)
e
.=ro 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm"')
Ritodrine Hydrochloride RS313 Instrumenl: Fourier Transform Phase: Potassium Chloride Disc
100,0 T"" '"'''''-''' ' ' ' ' ' ' ' - ' ' - - ' ' ' ' 7 - ''''''''' "'"''''''''''''''' ' ' - T ' ''''''-'''''''''-''''''''''''''' --'T""·"·,·,,·,"""""-"",,· ...,""""·"T"""""--",, ·,, """, .... "",,,,,,,,,,,,,,,,,,,, """"-".,, ...
60 +""."'" ..._.
1800 1600 1400 1200 1000 800 600 400,0

Wavenumber (cm"')
Salbutamol RS314 Instrumen!: Dispersive Phase: Potassium Bromide Disc

-_. r------
.--
40 J
I
I I
;g
o
Cl)
ü 20
e
:§
-E I
(f)
e
ro
.= 0.0 I
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Salbutamol Sulfate RS3IS Instrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
¡
... }
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Salicylic Acid RS316 Instrumen!: Dispersive Phase: Potassium Bromide Disc

100.0
80
60 t
40 .1.,
i
B
e
20
ro
==
'E
(f)
e
i
ro I
.= 0.0 t--------- j
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm" )
Selegiline RS400 Instrument: Fourier Transform Phase: Thin Film

100,0 "
80
60 ¡
f
¡
¡
40
~ 20
e
ro
:t:::
E
en
e
ro
~
2000,0 1800 1600 1400 1200 1000 800 600 400,0
Wavenumber (cm-')
Sertraline Hydrochloride (A) RS460 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100,0
80
60 .
40
;ji!.
u
e
ID
20
ro
:t:::
E
en
e
~
t- 0,0 L ...
¡
,}
2000,0 1800 1600 1400 1200 1000 800 600 400,0

1
Wavenumber (cm- )
Sertraline Hydrochloride (B) RS443 Instrument: Fourier Transform Phase: 1% w/v solution in dichloromethane Thickness : 0,5 mm
100,0
70 .~ . _---~ .- ,J -~ .. - ,_.~ ._~.~._-
I
1
60 ,
.,
¡
50 ,
I
40 ¡ 1"
30
;ji!. "
ID
u 20
e
fl ,
'E 10 ., -¡ I
en
e
~
t- lO I ,'- ~ -' ~ ~_. -'---1- '" -t .t--
2000,0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm,1)
2014 Infrared Reference Spectra V-Sl2S
Simvastatin RS423 Inslrument: Fourier Transform Phase: POlassium Bromide Disc

100.0
-+-----.11. . . .
I
-1-....... - - - -...... - - - - - - - -.. - ...
2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm"')
Sodium Amidotrizoate RS317 Inslrumenl: Fourier Transform Phase: Polassium Bromide Disc
100.0 T ............................................. ...--,-..............................................., ................................................,......... ......................................... ........
80
?ft.
o
Q)
20
c:
ro
:::::
'ECf)
c:
ro
.= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Sodium Feredetate RS378 Inslrument: Fourier Transform Phase: Polassium Bromide Disc
100.0
60
~ 20 ·j_··_·_-·_···············- i-- · · ···. ·_~_ ··_ ~·f-·· ....................J- { ........... ............. :..
c:
ro
:::::
'E
Cf)
c:
ro I
.= 0.0 ..._.. ___.__ . ----l.--
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
v -S 126 Infrared Reference Spectra 20 14
Sod iu m Polystyrene Sulfonate RS3 18 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc
100.0 __
80
60
40
'i:ft.
Q)
ü 20
e
ro
:1='
E
en
e
ro
.= 0.0 ... __ .. +
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Spironolactone RS32 1 Instrumenl: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0. 1mm
100.0
!"
60 +______ ._..
40
'i:ft.
Q)
ü 20
e
ro
:1='
'E
en
e
.=ro 0.0 j------'---'--
2000.0 1800 1600 1400 .1200 1000 800 600 400.0
Wavenumber (cm-')
Stanozolol RS322 Instrumenl: Dispersive Phase: Potassium Bromide Disc

100.0
60
40
¡:s 20
e
ro
:1='
'E
en
e
ro
.= 0.0 +. . ,. 4 -+- -- .
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Sulfadiazine RS325 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
~
ID
u 20
c:
ro
::=
"E
en
c:
ro
r= 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-1)
Sulfamethoxazole RS327 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
'<f!.
ID
u 20
c:
ro
::=
"E
en
c:
~
1- 0"0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm- 1
)
Sulfasalazine RS429 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
60 ¡ ..........................
~ 20
c:
ro
=1='
"E
en
ffi
~ 0.0
2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Sulindac RS323 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 T' • ................... .... ............. : .......... .
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40
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Wavenumber (cm-')
Sulpiride RS391 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
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Sumatriptan RS414 Instrument: Fourier Transform Phase: Liquid Paraffin Mull

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Wavenumber (cm- ) 1
20 14 Infrared Reference Spectra V-S 129
Sumatriptan Succinate RS413 Instrument: Fourier Transform Phase: Liquid Paraffin Mull
100.0
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40
20
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Tamoxifen RS328 Instru ment: Dispersive Phase : Potassium Bromide Disc

100.0
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40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Terfenadine RS392 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
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m
c:
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Testosterone RS329 Instrument: Dispersive Phase: 5% w/v Solution in Chloroform Thickness: 0.1mm
100.0
80
60
40
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Testosterone Decanoate RS330 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
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Testosterone Isocaproate RS331 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Testosterone Propionate RS332 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Theophylline RS333 Instrument: Dispersive Phase: Liquid Paraffin Mull

100.0
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Thiopental RS334 Instnument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Thioridazine (1) RS335 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0 .. .. ,... ............. . .
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Thioridazine (2) RS336 Instrument: Dispersive Phase: Thin Film

100.0
80
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Thiotepa RS337 Instrument: Dispersive Phase: 2% w/v Solution in Carbon Disulphide Thickness: 0.1mm
100.0 T~~··~·_····_··~~-~_·· ...............,..... . - ........ _ ... .,~~-_.- ~"T '
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2000.0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Ticarcillin RS458 Instrument: Fourier Transform Phase : Potassium Bromide Disc

100.0
80
60
~ 20
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm,1)
Timolol RS339 Instrument: Dispersive Phase: Thin Film
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2000,0 1800 1600 1400 1200 1000 800 600 400.0
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Tioconazole RS393 Instrument: Fourier Transform Phase: Potassium Bromide Disc
80
60
40 + _____...."...""
2000,0 1800 1600 1400 1200 1000 800 600 400.0

Wavenumber (cm-')
Tioguanine RS340 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0 .
80
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
Tolazamide RS342 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
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60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Tolbutamide RS343 Instrument: Dispersive Phase : Potassium Bromide Disc

100.0
80
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Wavenumber (cm-')
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Tramadol Hyd rochloride RS465 Instrument: F ourier Transform Phase: Potassium Bromide Disc
100.0
80
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Trandolapril RS468 Instrument: Fourier Transform Phase: Attenuated Total Reflectance

100.0
80
60
40
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2000 _0 1800 1600 1400 1200 1000 800 600 400.0
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Tranexamic Acid RS344 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
80
60
40
2l 20
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(f)
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Tranylcyprornine Sulfate RS345 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
60 l.
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Trazodone Hydrochloride RS346 Instrument: Fourier Transform Phase: Potassium Chloride Disc
100.0
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Triarncinolone Acetonide RS348 Instrument: Fourier Transfomn Phase: Potassium Bromide Disc
100.0
80
60
25 20
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
Wavenumber (cm-')
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Triarncinolone Hexacetonide RS394 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
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Triclofos Sodium RS350 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0 ""
I
80
60
40
~ 20
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Trifluoperazine RS351 Instrument: Dispersive Phase: Thin Film

100.0
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Trifluridine RS469 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc
80
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Trimethoprim RS354 Instrumenl: Dispersive Phase: Potassium Bromide Disc

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Trimipramine Maleate RS355 Instrumenl: Dispersive Phase: Potassium Bromide Disc

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Triprolidine Hydrochloride RS356 Instrument: Dispersive Phase: Potassium Chloride Disc

100.0
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Tropicamide RS357 Instrument: Dispersive Phase: Potassium Bromide Disc

100.0
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40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Ursodeoxycholic Acid RS402 Instrument: Fourier Transform Phase: Potassium Bromide Disc
100.0
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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1000 I - Valproic Acid RS431 Instrument: Fourier Transform Phase: Thin Film
80 1--- ----- -!
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2000,0 1800 1600 1400 1200 1000 800 600 400,0
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Venlafaxine Hydrochloride RS439 Inslrument: Fourier Transform Phase: POlassium Chloride Disc
100.0 .,--__ --¡-----
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Verapamil RS359 Instrument: Dispersive Phase: Thin Film

100.0
80
40
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Vigabatrin RS360 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Warfarin RS361 Instrument: Dispersive Phase: Potassium Bromide Disc
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Xylometazoline RS362 Instrument: Dispersive Phase: 10% w/v Solution in Dichloromethane Thickness : O,1mm
100.0
80 ...
60
40 •._ __
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Zidovudine RS447 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Zopiclone RS430 Instrument: Fourier Transform Phase: Potassium Bromide Disc

100.0
I
80
60
40
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Zuclopenthixol Acetate RS363 Instrument: Fourier Transform Phase: Thin Film

100.0
80 +..
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40
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Zuclopenthixol Hydrochlo.-ide RS365 Inslrument: Dispersive Phase : Polassium Chloride Disc

100.0 .
80
60
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2000.0 1800 1600 1400 1200 1000 800 600 400.0
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Appendices
When a method, test or other matter described in an appendix is invoked in a monograph
reproduced from the European Pharmacopoeia, Part IJI of the General Notices applies.
When a method, test or other matter described in an appendix is invoked in any other
monograph, Part JI of General Notices applies.
2014 V-A3
Contents of the Appendices

EUROPEAN PHARMACOPOEIA EQUIVALENT TEXTS A13
APPENDIX I
A. General Reagents A19
B. Volumetric Reagents and Solutions A139
C. Standard Solutions A145
D. Buffer Solutions A150
E. Reference Materials A157
F. Polymorphism A157
APPENDIX II
A. Infrared Spectrophotometry A159
B. Ultraviolet and Visible Absorption Spectrophotometry A165
C. Nuclear Magnetic Resonance Spectrometry A166
D. Atomic Spectrophotometry: Emission and Absorption A170
E. Fluorescence Spectrophotometry [Fluorimetry] A175
F. X-Ray Fluorescence Spectrometry A175
G. Mass Spectometry A176
1. Inductive1y Coupled Plasma-Mas s Spectrometry A178
H. Raman Spectrometry A180
J. Flow Cytometry A181
K. Peptide Identification by Nuclear Magnetic Resonance Spectrometry A183
APPENDIX In
Chromatographic Separation Techniques A184
A. Thin-Iayer Chromatography A191
B. Gas Chromatography A194
C. Size-exclusion Chromatography A196
D. Liquid Chromatography A199
E. Paper Chromatography A200
F. Electrophoresis A201
G. Capillary Electrophoresis A206
H. Supercritical Fluid Chromatography A212
J. Isoe1ectric Focusing A212
K. Peptide Mapping A214
L. Amino Acid Analysis A217
M. Glycan Analysis of Glycoproteins A225
APPENDIX IV
A. Clarity of Solution A231
B. Colour of Solution A233
APPENDIX V
Determination of:
A. Melting Point A235
B. Freezing Point A239
C. Distillation Range A239
V-A4 2014
D. Boiling Point A240

E. Refractive Index A241
F. Optical Rotation and Specific Optical Rotation A241
G. Weight per Millilitre, Density, Relative Density and Apparent Density A241
H. Viscosity A243
J. Circular Dichroism A247
K. Relationship Between Reaction of Solution, Approximate pH and
Colour of Certain Indicators A248
L. Determination of pH Values A248
M. Thermal Analysis A250
N. Osmolality A252
O. Conductivity A252
P. Total Organic Carbon in Water for Pharmaceutical Use A253
Q. Density of Solids A254
APPENDIX VI
Qualitative Reactions and Tests A255
APPENDIX VII
Nessler Cylinders A259
Tubes for Comparative Tests A259
Limit Test for:
Aluminium A259
Ammonium A259
Arsenic A259
Calcium A260
Chlorides A260
Fluorides A260
Heavy Metals A261
Iron A264
Lead in Sugars A264
Magnesium A264
Magnesium and Alkaline-earth Metals A264
Heavy Metals in Herbal Drugs and Fatty Oils A264
Nickel in Polyols A265
Phosphates A265
Potassium A265
Sulfates A266
APPENDIX VIII
A. Non-aqueous Titration A267
B. Amperometric, Potentiometric and Voltametric Titrations A267
C. Oxygen-flask Combustion A268
D. Complexometric Titrations A268
E. Potentiometric Determination of Ionic Concentration using Ion-selective
Electrodes A269
F. Determination of Ethanol A270
2014 V-A5
G. Determination of Methanol and Propan-2-ol A273

H. Determination of Nitrogen A274
J. Tetrazolium Assay of Steroids A275
K. Ethylene Glycol and Diethylene Glycol in Ethoxylated Substances A275
L. Residual Solvents A276
M. Residual Ethylene Oxide and Dioxan A281
N. N,N-Dimethylaniline A282
O. 2-Ethylhexanoic Acid A282
P . Total Protein A283
Q. Acetic Acid in Synthetic Peptides A286
R. Nickel in Hydrogenated Vegetable Oils A286
S. Methyl, Ethyl and Isopropyl Methanesulfonate in Methanesulfonic Acid A287
T. Methyl, Ethyl and Isopropyl Methanesulfonate in Active Substances A288
V. Methanesulfonyl Chloride in Methanesulfonic Acid A289
W. Determination of Metal Catalyst or Metal Reagent Residues A290
APPENDIX IX
Determination of:
A. Sulfated Ash A293
B. Sulfur Dioxide A293
C. Water A294
D. Loss on Drying A296
E. Limit Test for Carbon Monoxide in Medicinal Gases A296
F. Carbon Dioxide in Medicinal Gases A297
G. Nitrogen Monoxide and Nitrogen Dioxide in Medicinal Gases A298
H. Oxygen in Medicinal Gases A298
J. Water in Medicinal Gases A298
K. Gas Detector Tubes A299
L. Nitrous Oxide in Gases A299
M. Water-Solid Interactions: Determination of Sorption-Desorption
Isotherms and ofWater Activity A300
APPENDIX X
A. Acetyl Value A304
B. Acid Value A304
C. Ester Value A304
D. Hydroxyl Value A304
E. Iodine Value A305
F. Peroxide Value A306
G. Saponification Value A306
H. Unsaponifiable Matter A307
J. Determination of Cineole A307
K. Determination of Aldehydes A308
L. Oxidising Substances A308
M . Essential Oils A308
N. Fixed Oils A309
V-A6 2014
O. Anisidine Value A313

P. Oils Rich in Omega-3-acids A3l3
1. Composition of Fatty Acids A313
2. Total Cholesterol in Oils Rich in Omega-3-Acids A3l5
Q Sterols in Fatty Oils A316
APPENDIX XI
A. Total Solids A319
Bl. Ethanol-soluble Extractive A319
B2. Water-soluble Extractive A319
C. Swelling Index A319
D. Foreign Matter A319
E. Essential Oils in Herbal Drugs A319
F. Continuous Extraction of Drugs A320
G. Complete Extraction of Alkaloids A32l
H. Stomata A32l
J. Ash A322
K. Acid-insoluble Ash A322
L. Pesticide Residues A322
M. Tannins in Herbal Drugs A324
N. Bittemess Value A324
O. Vacant
P. Dry Residue of Extracts A325
Q. Loss on Drying of Extracts A325
R. Test for Aristolochic Acids in Herbal Drugs A325
l. Test for Aristolochic Acids in Herbal Drugs A325
2. Test for Aristolochic Acids 1 and II in Herbal Drugs A327
S. Determination of Mycotoxins in Herbal Drugs A327
l . Determination of Aflatoxin Bl in Herbal Drugs A327
2. Determination of Ochratoxin A in Herbal Drugs A329
T. Herbal Drugs: Sampling and Sample Preparation A330
U. Microscopic Examination of Herbal Drugs A33l
APPENDIX XII
A. Disintegration A333
l. Disintegration Test for Tablets and Capsules A333
2. Disintegration Test for Suppositories and Pessaries A335
B. Dissolution A336
1. Dissolution Test for Tablets and Capsules (Dissolution Test for
Solid Dosage Forms) A336
2. Dissolution Test for Transdermal Patches A346
3. Dissolution Test for Lipophilic Solid Dosage Forms A349
4. Drug Release from Medicated Chewing Gum A350
5. Intrinsic Dissolution A354
6. Apparent Dissolution A356
2014 V-A7
C. Consistency of Formulated Preparations A357

1. Uniformity of Weight (Mass) A357
2. Uniformity of Weight (Mass) of Delivered Doses from Multidose
Containers A357
3. Uniformity of Content A357
4. Uniformity of Dosage Units A358
5. Extractable Volume of Parenteral Preparations A360
6. Vacant
7. Aerodynamic Assessment of Fine Particles - Fine Particle Dose and
Particle Size Distribution A361
8. Preparations for Nebulisation: Characterisation A373
9. Demonstration of Uniformity of Dosage Units Using Larger Sample
Sizes A376
APPENDIX XIII
Particulate Contamination A379
A. Sub-visible Particles A379
B. Visible Particles A381
APPENDIX XIV
Biological Assays and Tests A382
A. Microbiological Assay of Antibiotics A382
B. Immunochemical Methods A388
C. Test for Bacterial Endotoxins A390
D. Test for Pyrogens A394
E. Test for Abnormal Toxicity A395
F . Test for Depressor Substances A395
G. Test for Histamine A396
H. Monocyte-Activation Test A396
1. Vacant
J. Blood and Related Products A402
Coagulants A402
Al. Assay of Human Coagulation Factor II A402
A2. Assay of Factor VII Fraction A402
A3 . Assay of Factor VIII Fraction A403
A4. Assay of Factor IX Fraction A405
A5. Assay of Human Coagulation Factor X A405
A6. Assay of Human Coagulation Factor XI A405
A7. Activated Coagulation Factors A406
A8. Assay of Human von Willebrand factor A406
A9. Test for Prekallikrein Activator A407
AlO . Assay of Human Plasmin Inhibitor A408
Anticoagulants A408
Bl. Assay of Heparin in Coagulation Factors A408
B2. Assay of Heparin A409
B3. Assay of Human Antithrombin III A4l0
B4. Assay of Human Protein C A4l0
V-A8 2014
B5. Assay of Human Protein S A411

Immunoglobulins A412
Cl. Test for Fc Function ofImmunoglobulins A412
C2. Test for Anticomplementary Activity ofImmunoglobulin A413
C3. Assay of Human anti-D Immunoglobulin A415
C4. Test for anti-D Antibodies in Human Immunoglobulin A417
C5. Anti-A and anti-B Haemagglutinins A418
Other blood-related components A419
D 1. Assay of Human cx-l-Proteinase Inhibitor A419
K. Immunological Products A420
1. Assay of Diphtheria Vaccine (Adsorbed) A420
2. Assay of Pertussis Vaccine (Whole Cell) A424
3. Assay ofTetanus Vaccine (Adsorbed) A424
4. Assay of Hepatitis A Vaccine A429
5. Assay of Hepatitis B Vaccine (rDNA) A429
6. Assay of Pertussis Vaccine (Acellular) A430
7. In vivo Assay of Poliomyelitis Vaccine (Inactivated) A432
8. Flocculation Value (Lf) of Diphtheria and Tetanus Toxins and
Toxoids (Ramon Assay) A433
9. Residual Pertussis Toxin and Irreversibility of Pertussis A434
L. Nucleic Acid Amplification Techniques A434
M. Assay of Interferons A439
NI. Numeration of CD34/CD45+ Cells in Haematopoietic Products A441
N2. Colony-forming Cell Assay for Human Haematopoietic Progenitor Cells A443
N3. Nucleated Cell Count and Viability A444
APPENDIXXV
Production and Testing of Vaccines A446
A. Terminology used in Monographs on Biological Products A446
B. Aluminium in Adsorbed Vaccines A446
C. Ca1cium in Adsorbed Vaccines A447
D. Free Formaldehyde A447
E. Phenol in Immunosera (Antisera) and Vaccines A447
F. Neurovirulence A447
1. Test for Neurovirulence ofLive Virus Vaccines A447
2. Test for Neurovirulence of Poliomyelitis Vaccine (Oral) A448
G. Composition of Polysaccharide Vaccines A449
H. Chicken Flocks Free From Specified Pathogens for the Production and
Quality Control of Vaccines A452
J. Cell Substrates for the Production of Vaccines for Human Use A454
APPENDIX XVI
A. Test for Sterility A458
B. Microbiological Examination of Non-sterile Products A461
1. Test for Specified Micro-organisms A461
2. Microbial Enumeration Tests A466
2014 V-A9
3. Test for Absence of Mycoplasmas A470

4. Mycobacteria A475
5. Extraneous Agents in Viral Vaccines A475
C. Efficacy of Antimicrobial Preservation A476
D. Microbiological Quality of Non-sterile Phannaceutical Preparations and
Substances for Phannaceutical U se A478
E. Microbiological Control of Cellular Products A479
F. Microbiological Examination of Herbal Medicinal Products for Oral Use A480
G. Microbiological Quality of Herbal Medicinal Products for Oral Use A48l
APPENDIX XVII
A. Particle Size of Powders A483
l. Particle Size Classification of Powders A483
2. Powder Fineness A483
B. Sieves and Filters A483
l. Sieves A483
2. Filters A484
3. Particle-size Distribution Estimation by Analytical Sieving A485
C. Specific Surface Area by Air Permeability A487
D. Vacant
E. Flowability A489
F. Measurement of Consistency and Texture Analysis A490
l. Measurement of Consistency by Penetrometry A490
2. Texture Analysis of Semi-solids or Gels A492
G. Friability A493
l. Friability of Uncoated Tablets A493
2. Friability of Granules and Spheroids A493
H. Resistance to Crushing of Tablets A495
J. Softening Time Detennination of Lipophilic Suppositories A495
K. Pycnometric Density of Solids A496
L. Vacant
M. Specific Surface Area by Gas Adsorption A497
N. Powder Flow A500
O. Optical Microscopy A503
P. Particle Size Analysis by Laser Light Diffraction A504
Q. Characterisation of Crystalline and Partially Crystalline Solids by X-ray
Powder Diffraction (XRPD) A508
R. Porosity and Pore-size Distribution of Solids by Mercury Porosimetry A5l3
S. Bulk Density and Tapped Density of Powders A5l6
T. Wettability of Porous Solids Including Powders A5l8
U. Crystallinity A52l
V. Characterisation of Crystalline Solids by Microcalorimetry and Solution
Calorimetry A523
V-A10 2014
APPENDIX XVIII
Methods of Sterilisation A526
APPENDIX XIX
Containers A530
A. Introduction A530
B. Glass Containers for Pharmaceutical Use A530
C. Plastic Containers and Closures A535
1. Plastic Containers for Aqueous Solutions for Parenteral Infusions A536
D. Containers for Blood and Blood Components A537
1. Sterile Plastic Containers for Blood and Blood Components A537
2. Empty Sterile Containers of Plasticised Poly(Vinyl Chloride) for
Human Blood and Blood Components A539
3. Sterile Containers of Plasticised Poly(Vinyl Chloride) for Human
Blood Containing Anticoagulant Solution A539
E. Rubber Closures for Containers for Aqueous Parenteral Preparations A540
F. Sets for the Transfusion of Blood and Blood Components A54l
G. Sterile Single-use Plastic Syringes A543
APPENDIX xx
Materials U sed for the Manufacture of Containers A545
A. Materials for Containers for Human Blood and Blood Components A545
1. Materials Based on Plasticised Poly(Vinyl Chloride) for Containers
for Human Blood and Blood Components A545
2. Materials Based on Plasticised Poly(Vinyl Chloride) for Tubing
Used in Sets for the Transfusion of Blood and Blood Components A548
3. Materials Based on Non-plasticised Poly(Vinyl Chloride) for
Containers for Non-injectable, Aqueous Solutions A550
4. Materials Based on Non-plasticised Poly(Vinyl Chloride) for Dry
Dosage Forms for Oral Administration A552
5. Materials Based on Plasticised Poly(Vinyl Chloride) for Containers
for Aqueous Solutions for Intravenous Infusion A554
B. Polyolefines A557
C. Polyethylene A561
1. Polyethylene Without Additives for Containers for Parenteral
Preparations and for Ophthalmic Preparations A56l
2. Polyethylene With Additives for Containers for Parenteral and for
Ophthalmic Preparations A562
D. Polypropylene for Containers and Closures for Parenteral Preparations
and Ophthalmic Preparations A566
E. Poly(ethylene - vinyl acetate) for Containers and Tubing for Total
Parenteral Nutrition Preparations A569
F. Silicone A57l
1. Silicone Oil U sed as a Lubricant A57l
2. Silicone Elastomer for Closures and Tubing A572
G. Plastic Additives A573
H. Polyethylene Terephthalate for Containers for Preparations not for
Parenteral Use A576
2014 V-A11
APPENDIX XXI
A. Abbreviated Titles A579
B. Approved Synonyms A579
C. Codes for Eye Drops in Single-dose Containers A591
APPENDIX XXII
A. Viral Safety A592
B. Minimising the Risk of Transmitting Animal Spongiform Encephalopathy
Agents via Human and Veterinary Medicinal Products A592
APPENDIX XXIII
Weights and Measures A607
B. Conversion Tables for Commonly Used Units A607
APPENDIX XXIV
Abbreviations A608
APPENDIX XXV
Names, Symbols and Atomic Weights of Elements A609
2014 European Pharmacopoeia Equivalent Texts V-A13
European Pharmacopoeia Equivalent Texts

In monographs reproduced from the European Pharmacopoeia the analytical
methods, tests and other supporting texts are invoked by means of the reference
number of the text in the General Chapters of the European Pharmacopoeia.
The table below lists the contents of the General Chapters of the European
Pharmacopoeia and gives the British Pharmacopoeia or British Pharmacopoeia
(Veterinary) equivalents. It is provided for information but it is emphasised that,
for texts of the European Pharmacopoeia, in cases of doubt or dispute the text
published by the Council of Europe is authoritative. Appendices of the British
Pharmacopoeia (Veterinary) are identified by inclusion of '(Vet), after the
Appendix letter, for example, Appendix XV J (Vet) 1.
Ph. Eur. Subject of text British Phannacopoeia reference

t
1 General Notices
2.1.1 Droppers Appendix 1 A
2.1.2 Sintered-glass Filters Appendix XVII B2
2.1.3 UV lamps Appendix III A
2.1.4 Sieves Appendix XVII B 1
2.1.5 Tubes for Comparative Tests Appendix VII
2.1.6 Gas detector tubes Appendix IX K
2.2.1 Clarity and Degree of Opalescence of Liquids Appendix IV A
2.2.2 Degree of Coloration of Liquids Appendix IV B
2.2.3 Potentiometric Determination of pH Appendix V L
2.2.4 Reaction of Solution, pH and Indicator Colour Appendix V K
2.2.5 Relative Density Appendix V G
2.2.6 Refractive Index Appendix V E
2.2.7 Optical Rotation Appendix V F
2.2.8 Viscosity Appendix V H
2.2.9 Capillary Visco meter Method Appendix V H, Method II
2.2.10 Rotating Viscometer Method Appendix V H, Method III
2.2.11 Distillation Range Appendix V C
2.2.12 Boiling Point Appendix V D
2.2.13 Water by Distillation Range Appendix IX C, Method II
2.2.14 Melting point: Capillary Method Appendix V A, Method 1
2.2.15 Melting point: Open Capillary Method Appendix V A, Method IV
2.2.16 Melting point: Instantaneous Method Appendix V A, Method V
2.2.17 Drop Point Appendix V A, Method III
2.2.18 Freezing Point Appendix V B
2.2.19 Amperometric Titration Appendix VIII B
2.2 .20 Potentiometric Titration Appendix VIII B
2.2.21 Fluorimetry Appendix II E
2.2.22 Atomic Emission Spectrometry Appendix II D
2.2.23 Atomic Absorption Spectrometry Appendix II D
2.2.24 Infrared Spectrophotometry Appendix II A
2.2 .25 Visible and Ultraviolet Spectrophotometry Appendix II B
2.2.26 Paper Chromatography Appendix III E
2.2.27 Thin-layer Chromatography Appendix III A
2.2.28 Gas Chromatography Appendix III B
2.2.29 Liquid Chromatography Appendix III D
2.2.30 Size-exclusion Chromatography Appendix III C
2.2.31 Electrophoresis Appendix III F
2.2.32 Loss on Drying Appendix IX D
2.2.33 Nuclear Magnetic Resonance Spectrometry Appendix II C
2.2.34 Thermogravimetry Appendix V M
2.2.35 Osmolality Appendix VN
2.2.36 Ion-selective Potentiometry Appendix VIII E
2.2.37 X-ray Fluorescent Spectrophotometry Appendix II F
2.2.38 Conductivity Appendix V O
2.2 .39 Molecular Mass Distribution in Dextrans Appendix III C
2.2.40 Near Infrared Spectrometry Appendix II A
t Reproduced in full as Part Ilf of the General Notices of ¡he British Pharrnacopoeia and British Pharrnacoporia (Veten·nary).
V-A14 European Pharmacopoeia Equivalent Texts 2014
2.2.41 Circular Dichroism Appendix V J

2.2.42 Density of Solids Appendix V Q
2.2.43 Mass Spectrometry Appendix II G
2.2.44 Total Organic Carbon in Water Appendix V P
2.2.45 Supercritical Fluid Chromatography Appendix III H
2.2.46 Chromatographic Separation Techniques Appendix III
2.2.47 Capillary Electrophoresis Appendix III G
2.2.48 • Raman Spectroscopy Appendix II H
2.2.49 Falling BaH Viscometer Method Appendix V H, Method IV
2.2.54 Isoelectric Focussing Appendix III J
2.2.55 Peptide Mapping Appendix III K
2.2.56 Amino Acid Analysis Appendix III L
2.2.57 Inductively Coupled Plasma-Atomic Emission Spectrometry Appendix II D
2.2.58 Inductively Coupled Plasma-Mass Spectrometry Appendix II G 1
2.2.59 Glycan Analysis of Glycoproteins Appendix III M
2.2.60 Melting Point: Instrumental Method Appendix V A, Method VI
2.2.61 Characterisation of CrystaHine Solids by Microcalorimetry and Solution Appendix XVII V
Calorimetry
2.2.64 Peptide Identification by Nuclear Magnetic Resonance Spectrometry Appendix II K
2.2.65 Voltametric Titration Appendix VIII B
2.3.1 Identification Reactions Appendix VI
2.3.2 Identification of Fatty Oils by TLC Appendix X N
2.3.3 Identification of Phenothiazines by TLC Appendix III A
2.3.4 Odour Appendix VI
Limit test for:
2.4.1 - Ammonium Appendix VII
2.4.2 - Arsenic Appendix VII
2.4.3 - Calcium Appendix VII
2.4.4 - Chlorides Appendix VII
2.4.5 - Fluorides Appendix VII
2.4.6 - Magnesium Appendix VII
2.4.7 - Magnesium and Alkaline-earth Metals Appendix VII
2.4.8 - Heavy Metals Appendix VII
2.4.9 - Iron Appendix VII
2.4.10 - Lead in Sugars Appendix VII
2.4 .11 - Phosphates Appendix VII
2.4.12 - Potassium Appendix VII
2.4.13 - Sulfate Appendix VII
2.4.14 Sulfated Ash Appendix IX A, Method II
2.4 .15 Limit test for Nickel in Polyols Appendix VII
2.4.16 Total Ash Appendix XI J, Method II
2.4.17 Limit test for Aluminium Appendix VII
2.4 .18 Free Formaldehyde Appendix XV D
2.4.19 Alkaline Impurities in Fatty Oils Appendix X N
2.4.20 Determination of Metal Catalyst or Metal Reagent Residues Appendix VIII W
2.4.21 Foreign Oils in Fatty Oils by TLC Appendix X N
2.4.22 Composition of Fatty Acids by Gas Chromatography Appendix X N
2.4.23 Sterols in Fatty Oils Appendix X Q
2.4.24 Residual Solvents Appendix VIII L
2.4.25 Residual Ethylene Oxide and Dioxan Appendix VIII M
2.4.26 N,N-Dimethylaniline Appendix VIII N
2.4.27 Heavy Metals in Herbal Drugs and Fatty Oils Appendix VII
2.4 .28 2-Ethylhexanoic acid Appendix VIII O
2.4 .29 Composition of Fatty Acids in Oils Rich in Omega-3-acids Appendix X PI
2.4.30 Ethylene Glycol and Diethylene Glycol in Ethoxylated Substances Appendix VIII K
2.4.31 Nickel in Hydrogenated Vegetable Oils Appendix VIII R
2.4.32 Total Cholesterol in Oils Rich in Omega-3 Acids Appendix X P2
2.5.1 Acid Value Appendix X B
2.5.2 Ester Value Appendix XC
2.5 .3 Hydroxyl Value Appendix X D
2.5 .4 Iodine Value Appendix X E
2.5 .5 Peroxide Value Appendix X F
2.5.6 Saponification Value Appendix X G, Method II
2.5.7 Unsaponifiable Matter Appendix X H, Method II
2.5.8 Assay of Primary Aromatic Amino Nitrogen Appendix VIII B
2.5 .9 Semi-micro Determination of Nitrogen by Sulfuric Acid Digestion Appendix VIII H
2.5 .10 Oxygen-flask Method Appendix VIII C

2.5.11 Complexometric Titrations Appendix VIII D
2.5 .12 Semi-micro Determination of Water Appendix IX C, Method 1
2.5 .13 Aluminium in Adsorbed Vaccines Appendix XV B
2.5.14 Calcium in Adsorbed Vaccines Appendix XV C
2.5.15 Phenol in Immunosera and Vaccines Appendix XV E
Polysaccharide Vaccines:
2.5.16 - Protein Appendix XV G
2.5.17 - Nuc1eic Acids Appendix XV G
2.5.18 - Phosphorus Appendix XV G
2.5 .19 - O-acetyl Appendix XV G
2.5.20 - Hexosamines Appendix XV G
2.5.21 - Methylpentoses • Appendix XV G
2.5.22 - Uronic Acids Appendix XV G
2.5.23 - Sialic Acid Appendix XV G
2.5.24 Carbon Dioxide in Medicinal Gases Appendix IX F
2.5 .25 Carbon Monoxide in Medicinal Gases Appendix IX E
2.5.26 Nitrogen Monoxide and Nitrogen Dioxide in Medicinal Gases Appendix IX G
2.5.27 Oxygen in Medicinal Gases Appendix IX H
2.5 .28 Water in Medicinal Gases Appendix IX J
2.5.29 Sulfur Dioxide Appendix IX B, Method II
2.5.30 Oxidising Substances Appendix XL
2.5 .3 1 Ribose in Polysaccharide Vaccines Appendix XV G
2.5.32 Micro Determination of Water Appendix IX C, Method III
2.5.33 Total Protein Assay Appendix VIII P
2.5.34 Acetic Acid in Synthetic Peptides Appendix VIII Q
2.5.35 Nitrous Oxide in Gases Appendix IX L
2.5.36 Anisidine value Appendix X O
2.5.37 Methyl, Ethyl and Isopropyl Methanesulfonate in Methanesulfonic Acid Appendix VIII S
2.5.38 Methyl, Ethyl and Isopropyl Methanesulfonate in Active Substances Appendix VIII T
2.5.39 Methanesulfonyl Chloride in Methanesulfonic Acid Appendix VIII V
2.6.1 Sterility Appendix XVI A
2.6.2 Mycobacteria Appendix XVI B4
2.6.3 Vacant
2.6.4 Vacant
2.6.5 Vacant
2.6 .6 Vacant
2.6.7 Mycoplasmas Appendix XVI B3 and
Appendix XVI B(Vet)3
2.6 .8 Pyrogens Appendix XIV D
2.6.9 Abnormal Toxicity Appendix XIV E
2.6.10 Histamine Appendix XIV G
2.6.11 Depressor Substances Appendix XIV F
2.6.12 Microbiological Examination of Non-sterile Products: Microbial Appendix XVI B2
Enumeration Tests
2.6.13 Microbiological Examination of Non-sterile Products: Test for Appendix XVI B 1
Specified Micro-organisms
2.6. 14 Bacterial Endotoxins Appendix XIV C
2.6.15 Prekallikrein Activator Appendix XIV J A9
2.6.16 Extraneous Agents in Viral Vaccines Appendix XVI B5
2.6.17 Anticomplementary Activity of Immunoglobulins Appendix XIV J C2
2.6.18 Neurovirulence of Live Viral Vaccines Appendix XV F1
2.6.19 Neurovirulence of Poliomyelitis Vaccine (Oral) Appendix XV F2
2.6.20 Anti-A and Anti-B Haemagglutinins Appendix XIV J C5
2.6 .21 Nuc1eic Acid Amplification Appendix XIV L
2.6.22 Activated Coagulation Factors Appendix XIV J A 7
2.6.24 Avian Viral Vaccines: Tests for Extraneous Agents in Seed Lots Appendix XVI B(Vet)4
2.6.25 Avian Live Virus Vaccines : Tests for Extraneous Agents in Batches of Appendix XVI B(Vet)5
Finished Product
2.6.26 Test for Anti-D Antibodies in Human Immunoglobulin Appendix XIV J C4
2.6.27 Microbiological Control of Cellular Products Appendix XVI E
2.6.30 Monocyte-Activation Test Appendix XIV H
2.6.31 Microbiological Examination of Herbal Medicinal Products for Oral Appendix XVI F
Use
2.6 .33 Residual Pertussis Toxin and Irreversibility of Pertussis Toxoid Appendix XIV K9
2.7.1 Immunochemical Methods Appendix XIV B
2.7.2 Microbiological Assay of Antibiotics Appendix XIV A

2.7.3 Vacant
2.7.4 Assay of Coagulation Factor VIII Appendix XIV J A3
2.7.5 Assay of Heparin Appendix XIV J B2
2.7.6 Assay of Diphtheria Vaccine (Adsorbed) Appendix XIV K1
2.7.7 Assay of Pertussis Vaccine (Whole Cel!) Appendix XIV K2
2.7.8 Assay of Tetanus Vaccine (Adsorbed) Appendix XIV K3
2.7.9 Fc Function of Irnmunoglobulins Appendix XIV J C 1
2.7 .10 Assay of Human Coagulation Factor VII Appendix XIV J A2
2.7 .11 Assay of Coagulation Factor IX Appendix XIV J A4
2.7.12 Assay of Heparin in Coagulation Factors Appendix XIV J B 1
2.7.13 Assay of Anti-D Irnmunoglobulin Appendix XIV J C3
2.7.14 Assay of Hepatitis A Vaccine Appendix XIV K4
2.7.15 Assay of Hepatitis B (~DNA) Vaccine Appendix XIV K5
2.7.16 Assay of Pertussis Vaccine (Acellular) Appendix XIV K6
2.7.17 Assay of Human Antithrombin III Appendix XIV J B3
2.7.18 Assay of Human Coagulation Factor II Appendix XIV J Al
2.7.19 Assay of Human Coagulation Factor X Appendix XIV J A5
2.7.20 In vivo Assay of Poliomyelitis Vaccine (Inactivated) Appendix XIV K7
2.7.21 Assay of Human von Willebrand factor Appendix XIV J A8
2.7.22 Assay of Human Coagulation Factor XI Appendix XIV J A6
2.7.23 Numeration of CD34/CD45+ Cells in Haematopoietic Products Appendix XIV N
2.7.24 Flow Cytometry Appendix II J
2.7.25 Assay of Human Plasmin Inhibitor Appendix XIV J A10
2.7.27 Flocculation Value (Lf) ofDiphtheria and Tetanus Toxins and Toxoids Appendix XIV K8
(Ramon Assay)
2.7.28 Colony-forming Cell Assay for Human Haematopoietic Progenitor Appendix XIV N2
Cells
2.7.29 Nucleated Cell Count and Viability Appendix XIV N3
2.7 .30 Assay of Human Protein C Appendix XIV J B4
2.7.31 Assay of Human Protein S Appendix XIV J B5
2.7.32 Assay of Human a-1-Proteinase Inhibitor Appendix XIV J DI
2.8.1 Ash Insoluble in Hydrochloric Acid Appendix XI K, Method II
2.8.2 Foreign Matter Appendix XI D
2.8.3 Stomata and Stomatal Index Appendix XI H
2.8.4 Swelling Index Appendix XI C
2.8.5 Water in Essential Oils AppendixXM
2.8.6 Foreign Esters AppendixXM
2.8.7 Fatty Oils and Resinified Volatile Oils AppendixXM
2.8.8 Odour and Taste of Volatile Oils AppendixXM
2.8.9 Residue on Evaporation AppendixX M
2.8.10 Solubility in Ethanol (Volatile Oils) AppendixX M
2.8.11 Determination of Cineole Appendix XJ
2.8.12 Essential Oil Content of Crude Drugs Appendix XI E
2.8.13 Pesticide Residues Appendix XI L
2.8.14 Determination of Tannins in Herbal Drugs Appendix XI M
2.8 .15 Bittemess value Appendix XI N
2.8.16 Dry Residue of Extracts Appendix XI P
2.8.17 Loss on Drying of Extracts Appendix XI Q
2.8.18 Determination of Aflatoxin Bl in Herbal Drugs Appendix XI SI
2.8.20 Herbal Drugs: Sampling and Sample Preparation Appendix XI T
2.8 .21 Test for Aristolochic Acids in Herbal Drugs Appendix XI R1
2.8.22 Determination of Ochratoxin A in Herbal Drugs Appendix XI S2
2.8.23 Microscopic Examination of Herbal Drugs Appendix XI U
2.9.1 Disintegration of Tablets and Capsules Appendix XII Al
2.9.2 Disintegration of Suppositories and Pessaries Appendix XII A2
2.9 .3 Dissolution Test for Solid Dosage Forms Appendix XII B 1
2.9 .4 Dissolution Test for Transdermal Patches Appendix XII B2
2.9.5 Uniformity of Mass Appendix XII C 1
2.9.6 Uniformity of Content Appendix XII C3
2.9.7 Friability of Uncoated Tablets Appendix XVII G 1
2.9.8 Resistance to Crushing of Tablets Appendix XVII H
2.9.9 Measurement of Consistency by Penetrometry Appendix XVII F
2.9.10 Ethanol Content Appendix VIII F, Method III
2.9.11 Methanol and 2-propanol Appendix VIII G
2.9.12 Particle Size Classification of Powders (Sieve Test) Appendix XVII Al
2.9.13 Vacant
2.9.14 Specific Surface Area by Gas Permeability Appendix XVII C
2.9 .15 Vacant
2.9.16 Flowability Appendix XVII E
2.9.17 Extractable Volume of Parenteral Preparations Appendix XII C5
2.9.18 Aerodynamic Assessment of Fine ParticIes Appendix XII C7
2.9.19 Particulate Contamination: Sub-visible ParticIes Appendix XIII A
2.9 .20 Particulate Contamination: Visible ParticIes Appendix XIII B
2.9 .21 Vacant
2.9.22 Softening Time of Lipophilic Suppositories Appendix XVI! J
2.9.23 Pycnometric Density of Solids Appendix XVII K
2.9.24 Vacant
2.9.25 Dissolution Test for Medicated Chewing Gums Appendix XII B4
2.9.26 Specific Surface Area By Gas Adsorption Appendix XVII M
2.9.27 Uniformity of Mass of Delivered Doses from Multidose Containers Appendix XII C2
2.9.28 Vacant
2.9.29 Intrinsic Dissolution Appendix XII B5
2.9.31 ParticIe Size Analysis by Laser Light Difftaction Appendix XVII P
2.9.32 Porosity and Pore-size Distribution of Solids by Mercury Porosimetry Appendix XVII R
2.9.33 Characterisation of CrystaIline and PartiaIly CrystaIline Solids by X-ray Appendix XVII Q
Powder Difftaction (XRPD)
2.9.34 Bulk Density and Tapped Density of Powders Appendix XVII S
2.9.35 Powder Fineness Appendix XVII A2
2.9.36 Powder Flow Appendix XVII N
2.9 .37 Optical Microscopy Appendix XVII O
2.9.38 ParticIe-size Distribution Estimation by Analytical Sieving Appendix XVII B3
2.9 .39 Water-Solid !nteractions: Determination of Sorption-Desorption Appendix IX M
Isotherms and ofWater Activity
2.9.40 Uniformity of Dosage Units Appendix XII C4
2.9.41 Friability of Granules and Spheroids Appendix XVII G2
2.9 .42 Dissolution Test for Lipophilic Solid Dosage Forms Appendix XII B3
2.9.43 Apparent Dissolution Appendix XII B6
2.9.44 Preparations for Nebulisation: Characterisation Appendix XII C8
2.9.45 Wettability of Porous Solids IncIuding Powders Appendix XVII T
2.9.46 Vacant
2.9.47 Demonstration ofUniformity ofDosage Units Using Larger Sample Appendix XII C9
Sizes
3.1.1 Material Used for Containers for Human Blood and Blood Appendix XX A
Components
3.1.1.1 Materials Based on PVC for BIood and Blood Components Appendix XX Al
3.1.1.2 Materials Based on PVC for Tubing Used in Sets for the Transfusion Appendix XX A2
of BIood and Blood Components
3.1.3 Polyolefines Appendix XX B
3.1.4 Polyethylene - Without Additives Appendix XX C 1
3.1.5 Polyethylene - With Additives Appendix XX C2
3.1.6 Polypropylene Appendix XX D
3.1.7 Poly(ethylene-vinyl acetate) Appendix XX E
3.1.8 Silicone Oil Used as a Lubricant Appendix XX F 1
3.1.9 Silicone Elastomer for Closures and Tubing Appendix XX F2
3.1.10 Materials Based on Non-plasticised PVC for Containers for Appendix XX A3
Non-injectable, Aqueous Solutions
3.1.11 Materials Based on Non-plasticised PVC for Containers for Dry Appendix XX A4
Dosage Forms for Oral Administration
3.1.12 Vacant
3.1.13 Plastic Additives Appendix XX G
3.1.14 Materials Based on Plasticised Poly(vinyl chloride) for Containers for Appendix XX A5
Aqueous Solutions for !ntravenous Infusion
3.1.15 Polyethylene Terephthalate for Containers for Preparations Not for Appendix XX H
Parenteral Use
3.2 Containers Appendix XIX A
3.2.1 Glass Containers for Pharmaceutical Use Appendix XIX B
3.2.2 Plastic Containers and Closures Appendix XIX C
3.2.2.1 Plastic Containers for Aqueous Solutions for Parenteral !nfusions Appendix XIX C 1
3.2.3 Sterile Plastic Containers for Blood and Blood Components Appendix XIX DI
3.2.4 Empty Sterile Plastic Containers for Blood and Blood Components Appendix XIX D2
3.2.5 Sterile Plastic Containers with Anticoagulant for Blood and Blood Appendix XIX D3
Components
3.2.6 Sets for the Transfusion of Blood and Blood Components Appendix XIX F
3.2.7 Vacant
3.2.8 Sterile Single-Use Plastic Syringes Appendix XIX G
3.2.9 Rubber Closures for Containers for Aqueous Preparations for Appendix XIX E
Parenteral Use
4 .1.1 Reagents Appendix I A
4.1.2 Standard Solutions Appendix I C
4.1.3 Buffer Solutions Appendix I D
4.2.1 Reference Substances for Volumetric Solutions Appendix I B
4.2.2 Volumetric Solutions Appendix I B
5.1.1 Methods of Preparation of Sterile Products Appendix XVIII
5.1.2 Biological Indicators (sterilisation) Appendix XVIII
5.1.3 Efficacy of Antimicrobial Preservation Appendix XVI C
5.1.4 Microbiological Quality of Non-sterile Pharmaceutical Preparations and Appendix XVI D
Substances for Pharmaceutical Use
5.1.5 Fo Concept (sterilisation) Appendix XVIII
5.1.6 Alternative Methods for Control of Microbiological Quality Supplementary Chapter IV L
5.1.7 Viral Safety Appendix XXII A
5.1.8 Microbiological Quality of Herbal Medicinal Products for Oral Use Appendix XVI G
5.1.9 Guidelines for using the Test for Sterility Supplementary Chapter IV P
5.1.10 Guidelines for using the test for bacterial endotoxins Supplementary Chapter I C
5.2.1 Terminology: Vaccines Appendix XV A and
Appendix XV Aevet)
5.2.2 SPF Chicken Flocks Appendix XV H and
Appendix XV Hevet)
5.2.3 Cell Substrates for the Production of Vaccines for Human Use Appendix XV J
5.2.4 Cell Cultures (Veterinary Vaccine Production) Appendix XV Jevet) 1
5.2.5 Substances of Animal Origin for the Production of Immunological Appendix XV Jevet)2
Veterinary Medicinal Products
5.2.6 Safety: Veterinary Vaccines Appendix XV Kevet) 1
5.2 .7 Efficacy: Veterinary Vaccines Appendix XV Kevet)2
5.2.8 Minimising the Risk of Transmitting Animal Spongiform Appendix XXII B
Encephalopathy Agents via Medicinal Products
5.2.9 Evaluation of Safety of Each Batch of Immunosera for Veterinary U se Appendix XV Kevet)3
5.3 Statistii::al Analysis of Biological Assays and Tests Supplementary Chapter IV G
5.4 Residual Solvents Supplementary Chapter IV D
5.5 Alcoholimetric Tables Supplementary Chapter IV E
5.6 Assay of Interferons Appendix XIV MI
5.7 Physical Characteristics of Radionuclides Radiopharmaceutical Preparations
5.8 Pharmacopoeial Harmonisation Supplementary Chapter IV F
5.9 Polymorphism Appendix I F
5.10 Control of Impurities in Substances for Pharmaceutical Use Supplementary Chapter IV J
5.11 Characters Section in Monographs Supplementary Chapter IV K
5.12 Reference Standards Supplementary Chapter IV M
5.14 Gene Transfer Medicinal Products for Human Use Supplementary Chapter IV N
5.16 Crystallinity Appendix XVII U
5.17.1 Recommendations on dissolution testing Supplementary Chapter I E
5.20 Metal Catalyst or Metal Reagent Residues Supplementary Chapter IV Q
2014 Appendix 1 A V-A19
5
Appendix 1
The specifications given below are strictly for the use of the
material s as reagents. The inclusion of a material in this
Appendix does not imply that it is suitable for use in
medicines. Exceptionally, a trademark or supplier may be
indicated for certain reagents whose availabiliry is limited.
This information is given only to make it easier to obtain
such reagents and this does not suggest in any way that the
mentioned suppliers are especially recommended or certified
by the European Pharmacopoeia Commission or the Council
of Europe. It is therefore acceptable to use reagents from
another source provided that they comply with the standards
of the Pharmacopoeia.
o
--
lt)
A. General Reagents 10
Where the name of substance or a solution is followed by the
letter R (the whole in italics), this indicates a reagent
included in the following listo The specifications given for
reagents do not necessarily guarantee their qualiry for use in
medicines.
The description may include a CAS number (Chemical
Abstract Service Registry Number) recognisable by its rypical e
format, for example (9002-93-1).
Some of the reagents included may be injurious to health
unless adequate precautions are taken. They should be
handled in accordance with good laboratory practice and any
relevant regulations such as those issued in the United
Kingdom in accordance with the Health and Safery at Work
-
a
Act (1974). 3.00- 3.05

Reagents in aqueous solution are prepared using water R.
Where a reagent solution is described using an expression Fig. 1-1 Standard dropper
such as "hydrochloric acid (10 giL HCl)", the solution is
Dimensions in millimetres
prepared by an appropriate dilution with water R of a more
concentrated reagent solution specified in this chapter.
Reagent solutions used in the limit tests for barium, calcium Acacia Of the British Pharmacopoeia.
and sulfates are prepared using distilled water R. Where the Acacia Solution Dissolve 100 g of acacia in 1000 mL of
name of the solvent is not stated, an aqueous solution is water, stir mechanically for 2 hours, centrifuge at 2000 g for
intended. 30 minutes to obtain a clear solution.
The reagents and reagent solutions are to be stored in well- Store in polyethylene conrainers of about 250 mL capaciry at
closed containers . The labelling should comply with the 0° to - 20°.
relevant nationallegislation and intemational agreements. Acebutolol Hydrochloride (34381-68-5)
Droppers Of the British Pharmacopoeia.
(Ph Eur text 2.1.1) Aceta! 1,I-Diethoxyethane C 6H 14 0 2 = 118.2 (105-57-7)
The term 'drops' means standard drops delivered from a General reagent grade of commerce. dig,
about 0.824;
standard dropper as described below. n~, about 1.382; boiling point, about 103°.
Standard droppers (Fig. 1-1) are constructed of practically Acetaldehyde C 2 H 4 0 = 44.1 (75-07-0)
colourless glass. The lower extremiry has a circular orifice in General reagent grade of commerce.
a fiat surface at right angles to the axis. Other droppers may
be used provided they comply with the following test.
A clear, colourless fiammable liquid; dig,about 0.788;
n~o, about 1.332; boiling point, about 21 °.
Twenry drops of water at 20 ± 1° fiowing freely from the
Acetaldehyde Anunonia Trimer Trihydrate
dropper held in the vertical position at a constant rate of one
2,4,6-Trimethylhexahydro-l,3,5-triazine trihydrate;
drop per second weighs 1000 ± 50 mg. The dropper must
C6H1 SN3,3H20 = 183.3 (76231-37-3)
be carefully cleaned before use. Carry out three
determinations on any given dropper. No result may deviate Melting point, 95° to 9r.
by more than 5% from the mean of the three determinations Acetanúde C 2H sNO = 59.07 (60-35-5)
General reagent grade of commerce.
Melting point, about 78°.
Acetic Acid Dilute 30 g of glacial acetic acid to 100 mL
with water. It contains not les s than 29.0% and not more
than 31.0% w/v of C 2H 40 2 (5M).
V -A20 Appendix 1 A 2014
Acetic Acid, Anhydrous Anhydrous glacial acetic acid; Acetoxyvalerenic Acid (2E)-3-[(lRS,4S,7R,7aR)-
C ZH 40z = 60.1 (64-19-7) 1-(Acetyloxy)-3, 7-dimethyl-2,4,5,6, 7, 7a-hexahydro-1H-inden-
Glacial acetic acid of commerce for use in non-aqueous 4- yl]-2-methylprop-2-enoic acid;
titrations containing not less than 99.6% w/w of C ZH 40 Z. C 17 H z4 0 4 = 292.4 (81397-67-3)
d~g, 1.0152 to 1.053; boiling point, 117° to 119°. Colourless or pale yellow viscous oil.
Complies with the following test. Complies with the following test.
Water Not more than 0.4% w/w, Appendix IX C. If the Absorbance (2.2.25) Absorption maximum at about
water content is greater than 0.4% it may be adjusted by 216 nm, determined in methanol.
adding the calculated amount of acetic anhydride. Acetyl Chloride C zH 3CIO = 78.5 (75-36-5)
Store protected from light. Analytical reagent grade of commerce.
Acetic Acid, Dilute Dilute 12 g of glacial acetic acid to d~g, about l.l O. Not less than 95% distils between 49° and
100 mL with water. It contains not less than 11.5% and not 53°.
more than 12.5% w/v of C Z H 40z (2M).
Acetylacetamide 3-0xobutanamide; C4H 7NO z = 101.1
Acetic Acid, Glacial (5977-14-0)
Analytical reagent grade of commerce. General reagent grade of commerce.
A colourless liquid, about 17. 5M in strength; freezing point, Melting point, 53° to 56°.
about 16°; weight per mL, about 1.05 g. It contains not les s
Acetylacetone Pentane-2,4-dione; CsHsO z = 100.1
than 98.0% w/w of C ZH 40z. (123-54-6)
Solutions of molarity XM should be prepared by diluting
Analytical reagent grade of commerce.
57x mL (60x g) of glacial acetic acid to 1000 mL with water.
A colourless or slightly yellow, easily ftammable liquido
Acetic Anhydride C 4H 60 3 = 102.1 (108-24-7)
n~o, 1.452 to 1.453; boiling point, 138 to 140°.
0
Analytical reagent grade of commerce containing not les s

than 97.0% w/v of C 4H 60 3. Acetylacetone Reagent Rl
0
A colourless liquid; boiling point, 136 to 142 0
•
To 100 mL of ammonium acetate solution add 0.2 mL of
acetylacetone.
Acetic Anhydride Solution Rl
Acetylacetone Reagent R2
Dissolve 25 mL of acetic anhydride in anhydrous pyridine and
dilute to 100 mL with anhydrous pyridine. Dissolve 0.2 mL of acetylacetone, 3 mL of glacial acetic acid
and 25 g of ammonium acetate in water and dilute to 100 mL
Store protected from light and airo
with the same solvento
Acetic Anhydride-Dioxan Solution Add 1 mL of acetic
4-Acetylbiphenyl 4-Phenylacetophenone;
anhydride to 50 mL of 1)4-dioxan.
C l4H 1Z O = 196.2 (51-42-3)
Acetic Anhydride-Sulfuric Acid Solution Acetic
Anhydride- Sulfuric Acid Solution Carefully mix 5 mL of
acetic anhydride with 5 mL of sulfuric acid. Add, dropwise and Melting point, about 117 0 •
with cooling, to 50 mL of absolute ethanol. N-Acetyl-E-caprolactam C sH 13N0 2 = 155.2 (1888-91-1)
Prepare immediately before use. General reagent grade of commerce.
Acetone Propan-2-one; C 3H 60 = 58.08 (67-64-1) d~g , about l.l00; n~, about 1.489; boiling point, about
Analytical reagent grade of commerce. 135°.
A volatile, ftammable liquid; boiling point, about 56°; weight Acetylcholine Chloride C 7 H l6 ClNO Z = 181.7 (60-31-1)
per mL, about 0.79 g. General reagent grade of commerce.
0
Complies with the following test. Store at -20 •
Water Not more than 0.3% w/w, Appendix IX C, using N-Acetyl-L-cysteine C sH 9 N0 3S = 163 .2 (616-91-1)
anhydrous pyridine as the solvent. General reagent grade of commerce.
Acetonitrile Methyl cyanide; CH 3CN = 41.05 (75-05-8) Melting point, about 110°; [a]~o, about +4.6.
General reagent grade of commerce. Acetyleugenol ClzHl403 = 206.2 (93-28-7)
A colourless liquid; d~g, about 0.78; n~o, about 1.344. Not General reagent grade of commerce.
less than 95% distils between 80° and 82°. A yellow, oily liquid; n~o, about 1.521; boiling point, 281 ° to
282°.
Acetonitrile used in spectrophotometry complies with the following
requirement. Acetyleugenol used in gas chromatography complies with the
following test.
Transmittance Not less than 98% in the range 255 to
420 nm using water in the reference cell, Appendix II B. Assay Carry out the test for Chromatographic profile
described under Clove Oil using the reagent being examined
Acetonitrile for Chromatography Chromatographic as the test solution. The area of the principal peak is not les s
grade of acetonitrile containing not less than 99.8% of than 98.0% of the total area of the peaks.
CzH3N that complies with the following test.
N-Acetylglucosamine 2-(Acetylamino)-2-deoxy-
Transmittance Not les s than 98% from 240 nm using D-glucopyranose; C SH 1SN0 6 = 221.2 (7512-17-6)
water in the reference cel!. Melting point, about 202 0
•
Acetonitrile Rl Acetonitrile containing not less than 99.9% Acetyl-ll-keto- ~-boswellic Acid 3cx-(Acetyloxy)-
of CzH3N and complying with the following requirement. II-oxours-12-en-24-oic acid. (4~)-3cx-(Acetyloxy)-11-oxours-
Light absorption The absorbance of the reagent being 12- en-23-oic acid; C3zH4S0S = 512.7 (67416-61-9)
examined at 200 nm, Appendix II B, is not more than 0.10. White or almost white powder, insoluble in water, soluble in
Use water in the reference cel!. acetone, in anhydrous ethanol and in methano!.
2014 Appendix 1 A V -A21
Melting point, 271 ° to 274°. Acid Blue 93 Solution

Aceryl-11-keto-f3-boswellic acid used in liquid chromatography Dissolve 0.2 g of acid blue 93 in water and dilute to 100 mL
complies with the following additional test. with the same solvent.
Assay Liquid chromatography (2.2.29) as prescribed in the Acrylarnide C 3H 5NO = 71.08 (79-06-1)
monograph on lndian Frankincense. General reagent grade of commerce.
Minimum 90 %, ca1culated by the normalisation procedure. Melting point, about 84°.
N-AcetyIneuraminic Acid O-Sialic acid; Acrylamidelbisacrylarnide (29:1) Solution, 30 per cent
CllHI 9NOg = 309.3 (131-48-6) Prepare a solution containing 290 g of acrylamide and 10 g
Melting point, about 186°, with decomposition; [alta, of methylenebisacrylamide per litre of water and filter.
about - 36 (1 % w/v in water). Acrylamidelbisacrylamide (36.5:1) Solution, 30 per cent
N-Acetyltryptophan CJ3H14N20 3 = 246 .3 (1218-34-4) Prepare a solution containing 292 g of acrylamide and 8 g of
General reagent grade of commerce. methylenebisacrylamide per litre of water and filter.
Melting point, about 205°. Acrylic Acid Prop-2-enoic acid; C 3H 40 2 = 72.1 (79-10-7)
Assay Dissolve 10 mg in sufficient of a mixture of General reagent grade of commerce containing not less than
10 volumes of acetonitrile and 90 volumes of water to produce 99% of C 3H 40 2 and stabilised with 0.02% of hydroquinone
100 mL. Examine as prescribed in the monograph for monomethyl ether.
Tryptophan under 1,1'-Ethylidenebis(tryptophan) and other lt polymerises readily in the presence of oxygen.
related substances. The area of the principal peak in the d~g, about 1.05; n~o, about 1.421; boiling point, about 141 °.
chromatogram obtained is not less than 99.0% of the areas Actein (23R,24R,25S,26S)-3~-(~-D-Xylopyranosyloxy)-
of all the peaks. 16~,23 :23,26:24,25-triepoxy-26-hydroxy-9, 19-cyclolanostan-
Acetyltyrosine Ethyl Ester Ethyl N-acetyl-L-tyrosinate; 12~-yl acetate; C37H56011 = 677 (18642-44-9)
C J3 H 17N0 4,H 20 = 269 .3 (36546-50-6) Acteoside 2-(3,4-Dihydroxyphenyl)ethyl 3-0-(6-deoxy-ex-L-
General reagent grade of commerce. mannopyranosyl)-4-0-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-
A white, crystalline powder; [al~, +21 to + 25 (1% w/v in enoyll-~-D-glucopyranoside, Verbascoside;
ethanol); A(1%, 1 cm) at 278 nm, 60 to 68 determined in C2gH36015 = 624.6 (61276-17-3)
ethano!. Light yellowish powder, freely soluble in water and in
Acetyltyrosine Ethyl Ester, 0.2M 0.2M Ethyl methanol.
acetyltyrosinate Melting point, about 140°, with decomposition.
Dissolve 0.54 g of aceryltyrosine ethyl ester in sufficient ethanol Adamantane Tricyclo[3.3.1.1 3,7ldecane;
(96%) to produce 10 mL. CIOHl 6 = 136.2 (281-23-2)
Acid Blue 83 CI 42660; Coomassie brilliant blue R250; Melting point, about 270°.
brilliant blue R; C4 5H4~3Na07S = 826 (6104-59-2) Adenine Of the British Pharmacopoeia.
General reagent grade of commerce. Adenosine 6-Amino-9-~-D-ribofuranosyl-9H-purine;
A brown powder. CIOHJ3N 50 4 = 267.2 (58-61-7)
Acid Blue 90 Cl 42655; Coomassie brilliant blue G; General reagent grade of commerce.
brilliant blue G; C47H48N3Na07S2 = 854 (6104-58-1) Melting point, about 234°.
A dark brown powder with a violet sheen; sorne particles Adipic Acid C 6H IO 0 4 = 146.1 (124-04-9)
have a metallic lustre.
General reagent grade of commence.
Complies with ¡he following tests. 0
Melting point, about 152 •
Light absorption A(l %, 1 cm) in a 0.001 % w/v solution
in phosphate buffer pH 7. O at 577 nm, greater than 500, Adrenaline (IR)-I-(3,4- Dihydroxyphenyl)-
Appendix II B, ca1culated with reference to the dried 2-(methylamino)ethano!. 4-[ (lR)-I-hydroxy-
substance. 2-(methylamino)ethyl]benzene-l ,2-diol;
C 9H 13 N0 3 = 183.2 (51-43-4)
Loss on drying When dried to constant weight at 100° to
105°, loses not more than 5.0% of its weight. General reagent grade of commerce.
Acid Blue 92 Coomassie blue; CI 13390; trisodium Adrenaline Acid Tartrate L-Epinephrine D-hydrogen
8-hydroxy-4'-phenylaminoazonaphthalene-3,5' ,6- tartrate; C J3 H 1gNO g = 333 (51-42-3)
trisulfonate; C26HI 6N3Na30lOS3 = 696 (3861-73-2) General reagent grade of commerce.
General reagent grade of commerce. Adrenalone Hydrochloride 1-(3,4-Dihydroxyphenyl)-
Dark blue crystals . 2-(methylamino )ethanone hydrochloride. 3' ,4'-Dihydroxy-
2-(methylamino) acetophenone hydrochloride;
Acid Blue 92 Solution Coomassie blue solution C 9H I2 CIN0 3 = 217.7 (62-13-5)
Dissolve 0.5 g of acid blue 92 in a mixture of 10 mL of glacial General reagent grade of commerce.
acetic acid, 45 mL of ethanol (96%) and 45 mL of water.
Aescin Escin; C54H84023,2H20 = 1138 (6805-41-0)
Acid Blue 93 Methyl Blue; Poirrier Blue; CI 42780;
mixture of triphenylrosaniline di- and tri sulfate and of A mixture of related saponins obtained from the seeds of
Aesculus hippocastanum L.
triphenylpararosaniline; C37H27N3Na209S3 = 800
(28983-65-4) A fine, almost white or slightly reddish or yellowish,
General reagent grade of commerce. amorphous powder.
Dark blue powder. Chromatography Examine as prescribed in the
monograph on Senega root (0202) but apply 20 IlL of the
Colour change pH 9.4 to pH 14.0. solution. After spraying with anisaldehyde solution R and
heating, the chromatogram shows a principal band with an Albumin, Bovine Bovine serum albumin (9048-46-8)
R p of about 0.4. Bovine serum albumin containing about 96% of protein.
Homogeneity Carry out the test for Identification White to light brownish-yellow powder.
described under Senega Root applying 20 ¡.¡L of solution (2) Water (2.5.12) Maximum 3.0%, determined on 0.800 g.
to the plateo After spraying with anisaldehyde solution the
Albumin, Bovine Rl (9048-46-8).
chromatogram exhibits a principal spot with an Rf value of
about 0.4. Bovine serum albumin containing about 96% of protein.
Aflatoxin B¡ (6aR,9aS)-4-Methoxy-2,3,6a,9a- White or light brownish-yellow powder.
tetrahydrocyclopenta[ cJfuro [3 f,2 f:4,5J furo [2,3- A1bumin, Human
h][lJbenzopyran-1,1l-dione; C 17H 1Z 0 6 = 312.3 (1162-65-8) Human serum albumin containing not less than 96% of
White or faint yellow crystals. albumino
Agar (9002-18-0) The dried extract from Gelidium sp. and Albumin Solution, Human Albumin (9048-46-8)
other algae belonging to the c1ass Rhodophyceae. Albumin Solution of the British Pharmacopoeia.
Microbiological reagent grade of commerce. Albumin Solution Rl, Human Albumin solution
Agarose for Chromatography (9012-36-6) Dilute human albumin solution with sufficient sodium chloride
Chromatographic reagent grade of commerce. injection to produce a protein concentration of 0.1 % w/vand
Swollen beads 60 ¡.¡m to 140 ¡.¡m in diameter presented as a adjust the pH to between 3.5 and 4.5 with glacial acetic acid.
4% suspension in water. It is used in size-exc1usion Alcohol See Ethanol (96%) .
chromatography for the separation of proteins with relative Alcohol, Aldehyde-free See Ethanol (96%), aldehyde-free.
molecular masses of 6 x 10 4 to 20 x 10 6 and of
Alcohol (x% v/v) See Ethanol (96%) .
polysaccharides with relative molecular mas ses of 3 x 10 3 to
5 x 10 6 . Aldehyde Dehydrogenase Enzyme obtained from baker's
yeast which oxidises acetaldehyde to acetic acid in the
Agarose for Chromatography, Cross-linked
presence of nicotinamide-adenine dinuc1eotide, potassium
(61970-08-9)
salts and thiols at pH 8.0.
Chromatographic reagent grade of commerce.
Prepared from agarose by reaction with 2,3-dibromopropanol
Aldehyde Dehydrogenase Solution Dissolve in water a
in strongly alkaline conditions.
quantity of aldehyde dehydrogenase equivalent to 70 units and
Swollen beads 60 ¡.¡m to 140 ¡.¡m in diameter presented as a dilute to 10 mL with the same solvento This solution is
4% suspension in water. It is used in size-exclusion stable for 8 hours at 4' .
chromatography for the separation of proteins with relative
Aldrin C 1zH s Cl 6 = 364.9 (309-00-2)
molecular masses of 6 x 10 4 to 20 x 10 6 and of
polysaccharides with relative molecular masses of 3 x 10 3 to Use a grade suitable for pesticide residue analysis. A suitable
5 x 10 6 • certified reference solution (10 ng/¡.¡l in cyc1ohexane) may be
used.
Agarose for Chromatography Rl, Cross-linked
( 65099-79-8) Boiling point, about 145°; melting point, about 104°.
Chromatographic reagent grade of commerce. Aleuritic Acid (9RS,10SR)-9,10,16-
Trihydroxyhexadecanoic acid; C16H3Z0S = 304.4 (533-87-9)
Prepared from agarose by reaction with 2,3-dibromopropanol
in strongly alkaline conditions. General reagent grade of commerce.
Swollen beads 60 ¡.¡m to 140 ¡.¡m in diameter presented as a Melting point, about 101 c •
4% suspension iri water. It is used in size-exc1usion Alizarin S CI 58005; alizarin red S;
chromatography for the separation of proteins with relative C14H7Na07S,H20 = 360.3 (130-22-3)
molecular mas ses of 7 x 10 4 to 40 x 10 6 and of General reagent grade of commerce.
polysaccharides with relative molecular masses of 1 x lOS to A yellowish brown ar orange-yellow powder.
2 X 10 7 .
Alizarin S Solution A O. l % w/v solution of alizarin S.
Agarose for Electrophoresis (9012-36-6)
Complies with the following tests.
Electrophoretic reagent grade of cornmerce.
Sensitivity to barium To 5 mL of 0.05M sulfun'c acid add
A neutral, linear polysaccharide, the main component of 5 mL of water, 50 mL of aceta te buffer pH 3. 7 and 0.5 mL of
which is derived from agar. the solution being examined. Add, dropwise, 0.05M barium
Agarose-DEAE for Ion Exchange Chromatography perchlorate VS. The colour changes from yellow to orange-
Chromatographic reagent grade of commerce. red.
Cross-linked agarose substituted with diethylaminoethyl Colour change pH 3.7 (yellow) to pH 5.2 (violet).
groups, presented as beads. Aluminium Al = 26.98 (7429-90-5)
Agarose/Cross-1inked Polyacry1amide Analytical reagent grade of commerce.
Agarose trapped within a cross-linked polyacrylamide A white, malleable, flexible, bluish metal available as bars,
network. It is used for the separation of globular proteins sheets, powder, strips or wire. In moist air an oxide film
with relative molecular masses of2 x 104 to 35 x 10 4 . forms which protects the metal from corrosion.
Alanine L-Alanine; C3H7NOZ = 89.1 (56-41-7) Aluminium Ch10ride Aluminium chloride hexahydrate;
General reagent grade of commerce. AlClJ ,6H20 = 24l.4 (7784-13-6)
Melting point, about 315°, with decomposition. General reagent grade of commerce containing not less than
~-Alanine See 3-Aminopropionic acid.
98.0% of AlCl J ,6H 2 0 .
Store in an airtight container.
Aluminium ChIoride Reagent Dissolve 2.0 g of Acidity or alkalinity pH of a suspension prepared by

aluminium ehlon"de in 100 mL of a 5% v/v solution of glacial shaking I g with 10 mL of carbon dioxide-free water, about
aeetie acid in methanol. 7.5, Appendix V L.
Aluminium ChIoride Solution Dissolve 65.0 g of Aluminium Oxide, Neutral AI 20 3 = 102.0
aluminium ehlmide in sufficient water to produce 100 mL, General reagent grade of commerce complying with the
add 0.5 g of aetivated ehareoal, stir for 10 minutes, filter and monograph for Dried Aluminium Hydroxide.
add to the filtra te, stirring continuously, sufficient of a Aluminium Potassium Sulfate Alum, aluminium
1% w/v solution of sodium hydroxide to adjust the pH to potassium sulfate, aluminium potassium sulfate
about 1.5 (approximately 60 mL is required). dodecahydrate, aluminium potassium sulfate dodecahydrate;
Aluminium Hydroxide Gel AI(OHh + aq (21645-51-2) AIK(S04)z,12H 20 = 474.4 (7784-24-9)
A hydrated grade of aluminium hydroxide of commerce. Analytical reagent grade of commerce.
Aluminium Nitrate AI(N0 3h9H 20 = 375.1 (7784-27-2) Aluminium Sulfate Aluminium sulfate;
Analytical reagent grade of commerce. AI 2 (S04)3,16H 20 = 630.4 (10043-01-3)
Deliquescent crystals. Analytical reagent grade of commerce.
Store in an airtight container. Allantoin C 4H 6 N 4 0 3 = 158.1 (97-59-6)
Aluminium Oxide, Activated Acid AI 20 3 = 102.1 Allantoin of the British Pharmacopoeia
An almost white, fine, granular powder, very hygroscopic, Amaranth S Cl 16185; acid red 27;
activated by heating at 200 0 to 250 D for 3 hours . C2oH¡¡N2Na301OS3 = 604 (915-67-3)
Mean particle size, 50 to 200 Jlm. General reagent grade of commerce.
Aluminium Oxide, Anhydrous AI 20 3 = 102.0 A deep brown or deep reddish brown, fine powder.
(1344-28-1) When used for the titration of iodine and iodides with
Use a grade of commerce consisting of y-A120 3 potassium iodate, the colour changes from orange-red to
dehydratedand activated by heat treatment. The particle size yellow.
is such thatit passes through a 150-Jlm sieve but is retained Amaranth Solution A 0.2% w/v solution of amaranth S.
on a 75-Jlmsieve. Americium-243 Spiking Solution
Aluminium Oxide, Basic A basic grade of aluminium Contains 50 Bq/L 243Pu and a 134 gIL solution of
oxide of commerce suitable for column chromatography. lanthanum ehloride heptahydrate in a 103 gIL solution of
Complies with the following test. hydroehlmic acid.
Alkalinity pH of a 10% w/v suspension in earbon dioxide- Amido Black lOB See Naphthalene black 12B
free water, shaken for 5 minutes, 9 to 10, Appendix V L.
Amido Black lOB Solution A 0.5% w/v solution of
W'hen used in monographs other chan chose of che European naphthalene blaek 12B in a mixrure of 10 volumes of acetic
Pharmaeopoeia, che following test applies. acid and 90 volumes of methanol.
Activity Pack into a chromatographic tube (25 cm x Aminoazobenzene IX-Aminoazobenzene;
10 mm) sufficient of the basic aluminium oxide to form a 4-aminoazobenzene; 4-phenylazoaniline; Cl 11000;
column 50 mm deep. Apply to the column a mixture of C¡2HllN3 = 197.2 (60-09-3)
5 mL of sudan yellow solucion and 5 mL of sudan red solution General reagent grade of commerce.
and elute with 20 mL of a mixrure of 1 volume of benzene
and 4 volumes of petroleum spirit (boiling range, 60° ro 80°) . Brownish yellow needles with a bluish tinge; melting point,
Sudan red forms a zone about 10 mm in depth on the upper about 128°.
part of the column separated by a colourless zone from a 2-Aminobenzoic Acid Anthranilic acid;
yellow zone of sudan yellow below. C 7 H 7 N0 2 = 137.1 (118-92-3)
Aluminium Oxide, Deactivated To a suitable basic General reagent grade of cornmerce.
alumina add 1.5 to 2 % of water, mix well and allow'to stand Melting point, about 145°.
ovemight in a stoppered bottle. The product complies with 3-Aminobenzoic Acid C 7 H 7 N0 2 = 137.1 (99-05-8)
the following test. White or almost white crystals. An aqueous solution tums
Prepare a column (20 cm x 10 mm) using the alumina and brown on standing in airo Melting point, about 174°.
hexane. Add a solution of 0.25 g of caleijerol in 10 mL of
Store in an airtight container, protected from light.
hexane. When the level of the solution falls just to the top of
the column, begin eluting with a 17.5% v/v solution of ether 4-Aminobenzoic Acid C 7 H 7 N0 2 = 137.1 (150-13-0)
in hexane adjusting the rate of flow, if necessary, to between General reagent grade of commerce.
1 and 2 mL per minute. Collect 200 mL of eluate; no Melting point, about 188°.
calciferol is present. Collect a further 100 mL of eluate; it Complies with the following test.
contains not les s than 95% of the ca1ciferol used in the test, Homogeneity Carry out the test for Related substances
when determined by the Assay described under Ca1ciferol described under Procaine Hydrochloride. The chromatogram
Oral Solution.
obtained shows only one principal spot.
Aluminium Oxide G A fine, white, homogeneous powder Store protected from Iight.
of an average particle size between 10 and 40 Jlm containing
about 10% w/w of ca1cium sulfate hemihydrate. 4-Aminobenzoic Acid Solution Dissolve 1 g of
4-aminobenzoic acid in a mixrure of 18 mL of anhydrous aeetic
Content of calcium sulfate Carry out the test described
aeid, 20 mL of water and 1 mL of orthophosphmic aeid.
under siliea gel G. lmmediately before use, mix 2 volumes of the solution with
3 volumes of acetone.
(4-Arninobenzoyl)-L-glutamic Acid 1- sulfonic acid. Dissolve 1.5 g of the mixture in water and
C12HI~20S = 266.2 (4271-30-1) dilute to 10 mL with the same solvento Prepare the solution
General reagent grade of commerce. daily.
Melting point, about 173°. Arninohydroxynaphthalenesulfonic Acid Solution,
Strong Aminohydroxynaphthalenesulphonic acid solution,
4-Aminobutanoic Acid See 4-Amino-n-butyric acid.
strong.
Aminobutanol See 2-Aminobutan-1-ol.
Dissolve 0.25 g of 4-amino-3-hydroxynaphthalene-1-sulfonic
2-Aminobutan-l-ol Aminobutanol; C 4 H II NO = 89.14 acid in 75 mL of a 15% w/v solution of sodium metabisulfite,
(5856-63-3) warming to assist solution if necessary. Add 2.5 mL of a
General reagent grade of commerce. 20% w/v solution of sodium sulfite, mix and add sufficient of
An oily liquid; boiling point, about 180°; d~g, about 0.94; the sodium metabisulfite solution to produce 100 mL.
n5°,about 1.453. 5-Aminoimidazole-4-carboxamide HydrochIoride
4-Amino-n-butyric Acid 4-Aminobutanoic acid; gamma C 4 H 6 N 4 0,HCI = 162.6 (72-40-2)
amino butyric acid; GABA; C 4H 9N0 2 = 103.1 (2835-81-6) General reagent grade of commerce.
General reagent grade of commerce containing not less than Melting point, about 251 °, with decomposition.
97% of C 4 H gN0 2. cis-Aminoindanol (1 S,2R)-I-Amino-2,3-dihydro-
AminochIorobenzophenone lH-inden-2-01; (-)-cis-l-Aminoindan-2-01;
See 2-Amino-5-chlorobenzophenone. C 9H II NO = 149.2 (126456-43-7)
2-Arnino-5-chIorobenzophenone Content, minimum 98.0% (sum of enantiomers, determined
Aminochlorobenzophenone; C 13H IO CINO = 23l.7 by gas chromatography).
(719-59-5)
[al~, - 69 to - 59, determined on a 2 gIL solution in
General reagent grade of commerce. chloroform; melting point, 118° to 122°.
A yellow, crystalline powder; melting point, about 97°. 3-Aminomethylalizarin-N,N-diacetic Acid
Complies with the following test. Aminomethylalizarindiacetic acid; alizarin complexone
Homogeneity Carry out the test for Related substances dihydrate; Cl gHISNOs,2H20 = 42l.4 (3952-78-1)
described under Chlordiazepoxide Hydrochloride applying to A fine, ochre to orange-brown powder; melting point, about
the plate 5 ~L of a 0.05% w/v solution in methanol. The 185°.
chromatogram shows only one spot with an Rf value of Complies with the following test.
about 0.9.
Loss on drying Not more than 10.0%, determined on
Store protected from light. 1 g.
4-Aminofolic Acid Aminopterine; Arninomethylalizarindiacetic Acid Reagent
(2S)-2- [[4- [[ (2,4-Diaminopteridin-6-
Solution I Dissolve 0.36 g of cerium(m) nitrate in sufficient
yl)methylJaminoJbenzoylJaminoJpentanedioic acid; N-[4-
water to produce 50 rnL.
[[ (2,4-Diaminopteridin-6-yl)methyIJ aminoJbenzoylJ-
Solution II Suspend 0.7 g of 3-aminomethylalizarin-
L-glutamic acid; CI9H20NsOs = 440.4 (54-62-6)
N,N-diacetic acid in 50 mL of water. Dissolve with the aid of
Yellowish powder; melting point, about 230°.
about 0.25 mL of 13.5M ammonia, add 0.25 mL of glacial
6-Aminohexanoic Acid 6-Aminocaproic acid; acetic acid and dilute to 100 mL with water.
C 6 H I3N0 2 = 13l.2 (60-32-2)
Solution III Dissolve 6 g of sodium aceta te in 50 mL of
General reagent grade of commerce. water, add 11.5 mL of glacial acetic acid and dilute to
Melting point, about 205°. 100 mL with water.
p-Arninohippuric Acid Aminohippuric acid; To 33 rriL of acetone add 6.8 mL of solution IlI, 1.0 mL of
N-( 4-aminobenzoyl)glycine; C 9H ION 20 3 = 194.2 (61-78-9) Sohltion II and 1.0 mL of solution 1 and dilute to 50 mL with
General reagent grade of commerce. water. Use within 5 days.
A white or almost whité powder; melting point, about 200°. Complies with the following test.
Arninohippurlc Acid Reagent Dissolve 3 g of phthalic Sensitivity To l. O mL of fiuoride standard solution
acid and 0.3 g of p-aminohippuric acid in sufficient ethanol (10 ppm F) add 19. O mL of water and 5. O mL of the reagent
(96%) to produce 100 mL. being examined. After 20 minutes, a distinct blue colour is
Arninohydroxynaphthalenesulfonic Acid produced.
See 4-Amino-3-hydroxynaphthalene-1-sulfonic acid. Arninomethylalizarindiacetic Acid Solution Dissolve
4-Amino-3-hydroxynaphthalene-l-sulfonic Acid 0.192 g of 3-aminomethylalizarin-N,N-diacetic acid in 6 mL of
Aminohydroxynaphthalenesulfonic acid; freshly prepared 1M sodium hydroxide. Add 750 rnL of water,
aminohydroxynaphthalenesulfonic acid; 4-Amino-3- 25 mL of succinate buffer solution pH 4.6 and, dropwise,
hydroxynaphthalene-l-sulfonic Acid C IOH 9N0 4 S = 239 .3 O.5M hydrochloric acid until the colour changes from violet-red
(116-63-2) to yellow (pH 4.5 to 5). Add 100 mL of acetone and dilute to
General reagent grade of commerce. 1000 mL with water.
A crystalline powder; melting point, about 300°, with 4-Aminomethylbenzoic Acid C SH 9N0 2 = 151 .2
decomposition. (56-91-7)
Store protected from light. General reagent grade of commerce.
Arninohydroxynaphthalenesulfonic Acid Solution 3-(Arninomethyl)pyridine (3-Pyridylmethyl)amine;
Aminohydroxynaphthalenesulphonic acid solution. 3-picolylamine; C 6H sN 2 = 108.1 (3731-52-0)
Mix 5.0 g of anhydrous sodium sulfite with 94.3 g of sodium General reagent grade of commerce.
hydrogensulfite and 0.7 g of 4-amino-3-hydroxynaphthalene-
2-Amino-5-methylthiazole See 5-Methylthiazol-2-ylamine. Aminopyrazo1one Solution See Aminophenazone soluciono

8-Aminonaphthalene-2-sulfonic Acid 8-Amino-2- 3-Aminosalicylic Acid 3-Amino-2-hydroxybenzoic acid;
naphthalenesulfonic acid; 8-Amino-2-naphthalenesulfonic C 7H 7N0 3 = 153.1 (570-23-0)
acid, l-naphthylamine-7-sulfonic acid, l-naphthylamine- Melting point, about 240 0
•
7-sulfonic acid, 8-aminonaphthalene-2-sulfonic acid; Slightly soluble in water.

C IO H 9N0 3 S = 223.2 (119-28-8)
4-Aminosalicylic Acid 4-Amino-2-hydroxybenzoic acid;
General reagent grade of commerce. C 7H 7 N0 3 = 153.1 (65-49-6)
Aminonaphthalenesulfonic Acid Solution White or almost white, bulky powder, slightly soluble in
Aminonaphthalenesulphonic acid solution. water, soluble in ethanol (96%), in dilute nitric acid and in
Mix 0.5 g of 8-aminonaphthalene-2-sulfonic acid, 30 mL of sodium hydroxide. Ir darkens on exposure to air and light.
glacial acetic acid and 120 mL of water and heat with stirring Melting point, 135 0 to 145°.
until dissolved. Allow to cool and filter.
Store at a temperature not exceeding 30", in an airtight
Use the solution within 3 weeks. container, protected from light.
Aminonitrobenzophenone Arnmonia NH 3 = 17.03
See 2-Amino-5-nitrobenzophenone.
For IBM and 13.5M ammonia use analytical reagent grade
2 -Amino-5-nitrobenzophenone solutions of commerce containing 35% and 25% w/w of NH 3
Aminonitrobenzophenone; C 13 HIONz0 3 = 242.2 and weighing 0.88 g and 0.91 g per mL, respectively.
(1775-95-7)
Solutions of molarity XM should be prepared by diluting
General reagent grade of commerce. 75x mL of 13.5M ammonia or 56x mL of IBM ammonia to
A yellow, crystalline powder; melting point, about 160°; 1000 mL with water.
A(l %, 1 cm), 690 to 720, determined at 233 nm When ammonia is specified and the strength is not stated, use
(0.001 % w/v in methanol). a reagent prepared by diluting 67 g of 13.5M ammonia to
4-Aminophenazone Aminopyrazolone; 4-aminoantipyrine; 100 mL with water. When used in the lirnit test for iron, it
4-amino-2,3-dimethyl-l-phenyl-5-pyrazolone; complies with the following test.
C¡¡H13N30 = 203 .3 (83-07-8) [ron Evaporate 5 mL of the reagent being examined to
General reagent grade of commerce. dryness on a water bath, add 10 mL of water, 2 mL of a
Light yellow needles or powder; melting point, about 108°. 20% w/v solution of cim'c acid and 0.1 mL of thioglycollic
Aminophenazone Solution Aminopyrazolone solution. acid. Add sufficient of the reagent to make the solution
alkaline and dilute to 20 mL with water. No pink colour is
A 0.1 % w/v solution of 4-aminophenazone in borate buffer
produced .
pH9.0.
Arnmonia, Chloride-free 13.5M ammonia that complies
2-Aminophenol CóH7NO = 109.1 (95-55-6)
with the following test.
Pale yellowish-brown crystals which rapidly become brown,
Chloride Evaporate 54 mL on a water bath almost to
sparingly soluble in water, soluble in alcohol. Melting point,
0 dryness and dilute to 15 mL with water. The solution
about 172 •
complies with the limit test for chlorides, Appendix VII
Store in an airtight container, protected from light. (1 ppm).
3-Aminophenol C 6H 7NO = 109.1 (591-27-5) Arnmonia, Concentrated Strong Ammonia Solution of
Pale yellowish-brown crystals, sparingly soluble in water. the British Pharmacopoeia (about 13.5M).
Melting point, about 122°. Arnmonia, Lead-free Complies with the requirements
4-Aminophenol C 6H 7NO = 109.1 (123-30-8) prescribed for dilute ammonia Rl and with the following
General reagent grade of commerce. additional test: to 20 mL of lead-free ammonia add 1 mL of
A white or slightly coloured, crystalline powder; melting lead-free potassium cyanide solution, dilute to 50 mL with water
point, about 186 0 , with decomposition. and add 0.10 mL of sodiwl1 sulfide solution. The solution is
not more intensely coloured than a reference solution
Contains not les s than 95 % CóH7NO.
prepared without sodium sulfide.
Store protected from light.
Arnmonia, Methanolic Solutions of the requisite molarity
Aminopolyether 4,7,13, 16,21,24-Hexaoxo-l, 10- may be obtained by diluting 13.5M ammonia or IBM ammonia
diazabicycJo[8,8,8)-hexacosane; C¡ SH 36NZOó = 376.5 with methanol as directed under ammonia.
(23978-09-8)
Ammonia Rl, Concentrated Concentrated ammonia
General reagent grade of commerce. containing not less than 32.0% w/w ofNH 3 (about 18M).
Melting point, 70° to 73°. dig, 0.883 to 0.889 .
3-Aminopropanol 3-Amino-l-propanol; 3-aminopropan- Store protected from atrnospheric carbon dioxide at a
1-01; C 3H 9 NO = 75 .1 (156-87-6) temperature below 20".
General reagent grade of commerce. Arnmonia Rl, Dilute Dilute 41 g of concentrated ammonia
Melting point, about 11 o; dig, about 0.99; I1~O , about l.46l. to 100 mL with water. The solution contains not less than
3-Aminopropionic Acid C3H 7NOZ = 89.1 (107-95-9) 10.0% and not more than 10.4% w/v of NH 3 (about 6M).
General reagent grade of commerce containing not less than Arnmonia RZ, Dilute Dilute 14 g of concentrated ammonia
99% of C3H 7NOZ' to 100 mL with water. The solution contains not less than
Melting point, about 200°, with decomposition. 3.3% and not more than 3.5% of NH 3 (about 2M).
Aminopterine See 4-Aminofolic acid. Arnmonia R3, Dilute Dilute 0.7 g of concentrated
ammonia to 100 mL with water. The solution contains not
Aminopyrazolone See 4-Aminophenazone.
less than 0.16 % w/v and not more than 0.18% w/v ofNH3 ·
V-A26 Appendix 1 A 2014
Ammonium Acetate C 2H 7N0 2 = 77.08 (631-61-8) Ammonium Dihydrogen Orthophosphate Ammoniurn

Analytical reagent grade of commerce. dihydrogen phosphate; (NH4)H 2P0 4 = 115 .0 (7722-76-1)
Store in an airtight container. Analytical reagent grade of commerce.
Ammonium Acetate Solution Dissolve 150 g of pH of a 2.3% w/v solution, about 4.2, Appendix V L.
ammonium acetate in water, add 3 mL of glacial acetic Ammonium Formate C 2 H sN0 2 = 63 .1 (540-69-2)
acid and dilute 10 1000 mL with water. General reagent grade of commerce.
Use within l week of preparation. Me1ting point, 119° to 121 0
•
Ammonium and Cerium Nitrate See Ammonium Store in an airtight container.

cerium(IV) nitrate.
Ammonium Hexafluorogermanate(rv)
Ammonium and Cerium Sulfate See Ammonium (NH 4)zGeF6 = 222.7 (16962-47-3)
cerium(IV) sulfate.
(lR)-(-)-Ammonium lO-Camphorsulfonate
Ammonium Hydrogen Carbonate Ammonium
10-Camphorsulfonic acid, 10-camphorsulphonic acid,
bicarbonate; NH4HC0 3 = 79.06 (1066-33-7)
ammoniurn salt, (IR)-(- )-Arnmonium 10-Camphorsulfonate;
CloHl904S = 249.3 (82509-30-6) Analytical reagent grade of commerce containing not less
than 99% of NH4HC0 3.
General reagent grade of commerce containing not less than
97.0% of (lR)-(- )-arnmonium camphorsulfonate; Ammonium Iron(m) Citrate Ferric ammonium citrate
(1185-57-5)
[al~, _18° ± 2° (5% w/v in water).
General reagent grade (brown) of commerce.
Ammonium Carbarnate Carbamic acid arnmonium salt;
CH6N 20 2 = 78.1 (1111-78-0) Ammonium Iron(n) Sulfate Ferrous ammonium sulfate;
ferrous ammonium sulphate; ammonium iron(n) sulphate;
Ammonium Carbonate (507-87-6) Ammonium hydrogen
(NH4 )zFe(S04)2,6H zO = 392.1 (7783-85-9)
carbonate and arnmonium carbamate in approximate1y
equimolecular proportions containing the equivalent of about Analytical reagent grade of commerce.
30% w/w of NH3. Store protected from light.
Analytical reagent grade of commerce. Ammonium Iron(m) Sulfate Ferric ammonium sulfate;
Store in a well-closed container at a temperature not fernc ammonium sulphate; ammonium iron(m) sulphate;
exceeding 20°. NH4 Fe(S04)z,12H 20 = 482.2 (7783-83-7)
Ammonium Carbonate Solution A 15.8% w/v solution Analytical reagent grade of commerce.
of ammonium carbonate. Ammonium Iron(m) Sulfate Solution Rl Ammonium
Ammonium carbonate solution Rl Dissolve 20 g of iron(m) sulphate solution Rl
ammonium carbonate R in 20 mL of dilute ammonia R1 and Dissolve 0.2 g of ammonium iron(m) sulfate in 50 mL of
dilute 10 100 mL with water R. water, add 5 mL of nitric acid and dilute 10 100 mL with
Ammonium Carbonate Solution, Dilute Dissolve 5 g of water.
ammonium carbonate in a mixture of 7.5 mL of 5M ammonia Ammonium Iron(m) Sulfate Solution R2 Ferric
and 50 mL of water, dilute to 100 mL with water and filter, if ammonium sulfate solution R2; ferric ammonium sulphate
necessary. solution R2; ammonium iron(m) sulfate solution R2
Ammonium Cerium(rv) Nitrate Ammonium and cerium A 10% w/v solution of ammonium iron(lll) sulfate. If
nitrate, ceric ammonium nitrate; (NH4)2[Ce(N03)6] = 548.2 necessary, filter before use.
(16774-21-3) Produces a deep red colour with arnmonium thiocyanate in
Analytical reagent grade of commerce. acid solutions.
Ammonium Cerium(rv) Sulfate Arnmonium and cerium Ammonium Iron(m) Sulfate Solution R5 Fernc
sulphate, ammonium and cerium sulfate, ceric arnmonium arnmonium sulfate solution R5; ferric ammonium sulphate
sulphate, ceric arnmonium sulfate, ammonium cerium(rv) solution R5; ammonium iron(m) sulphate solution R5
sulphate; 2(NH4)2S04,Ce(S04h2H20 = 632.6 Shake 30.0 g of ammonium iron(m) sulfate with 40 mL of
(18923-36-9) nitric acid and dilute to 100 mL with water. If the solution is
General reagent grade of commerce. turbid, it should be filtered or centrifuged.
Ammonium Chloride NH4CI = 53.49 (12125-02-9) Store protected from light.
Analytical reagent grade of commerce. Ammonium Iron(m) Sulfate Solution R6 Ferric
Ammonium Chloride Solution A 10.7% w/v solution of arnmonium sulfate solution R6; fernc ammonium sulphate
ammonium chloride. solution R6; arnmonium iron(m) sulphate solution R6
Ammonium Citrate Diarnmonium hydrogen citrate; Dissolve 20 g of ammonium iron(w) sulfate in 75 mL of water,
C6Hl4N207 = 226.2 (3012-65-5) add 10 mL of a 2.8% w/v solution of sulfuric acid (96% w/w)
General reagent grade of commerce. and dilute 10 100 mL with water.
Acidity pH of 2.26% w/v solution, about 4.3. Ammonium Mercaptoacetate Solution Add 300 mL of
Ammonium Citrate Solution Dissolve, with cooling, water 10 50 mL of mercaptoacetic acid, neutralise with about
500 g of citric acid in a mixture of 200 mL of water and 40 mL of 13.5M ammonia and dilute to 500 mL with water.
200 mL of 13.5M ammonia. Filter and dilute to 1000 mL Ammonium Mercurithiocyanate Reagent Dissolve 30 g
with water. of ammonium thiocyanate and 27 g of mercury(Il) chloride in
Ammonium Cobaltothiocyanate Solution Dissolve sufficient water to produce 1000 mL.
37.5 g of cobalt(Il) nitrate and 150 g of ammonium thiocyanate Ammonium Metavanadate Ammonium vana date;
in sufficient water to produce 1000 mL. NH 4V0 3 = 117.0 (7803-55-6)
Use within 1 day of preparation. Analytical reagent grade of commerce.
Anunonium Metavanadate Solution Dissolve, with Chloride 0.5 g complies with the limit test for chlorides,
heating, 5 g of anunonium metavanadate in a mixture of Appendix VII (lOO ppm).
10 mL of 5M sodium hydroxide and 90 mL of water, cool and Sulfate 1.0 g complies with the limit test for sulfates,
filter, if necessary, through glass wool. Appendix VII (150 ppm).
Anunonium Molybdate (NH4)6M07024,4H20 = 1236 Sulfated ash Not more than 0.05%, Appendix IX A,
(12054-85-2) Method n. Use 1 g.
Analytical reagent grade of commerce. Ammonium Oxalate (C0 2 NH 4h,H 20 = 142.1
Anunonium Molybdate Reagent Mix in the following (6009-70-7)
order 1 volume of a 2.5% w/v solution of ammonium Analytical reagent grade of commerce.
molybdate, 1 volume of a 10% w/v solution of L-ascorbic acid
Ammonium Oxalate Solution A 4% w/v solution of
and 1 volume of 3M sulfuric acid and add 2 volumes of water. ammonium oxalate.
Use within 1 day of preparation.
Ammonium Persulfate Arnmonium peroxodisulfate,
Anunonium Molybdate Reagent Rl Mix 10 mL of a ammonium peroxodisulphate, arnmonium persulphate;
6.0% w/v solution of disodium arsenate, 50 mL of ammonium (NH4)2S20S = 228.2 (7727-54-0)
nwlybdate solution, 90 mL of 1M sulfuric acid and add
sufficient water to produce 200 mL.
Ammonium Phosphate See Diammonium hydrogen
Condition the mixture at 37° for 24 hours and keep in amber orthophosphate.
fiasks.
Ammonium Polysulfide Solution Ammonium
Ammonium Molybdate Reagent R2 Dissolve 50 g of
polysulphide solution Dissolve a sufficient quantity of
ammonium molybdate R in 600 mL of water R . To 250 mL of
precipitated sulfur to produce a deep orange solution in a
cold water R add 150 mL of sulfuric acid R and cool. Mix the solution prepared in the following manner. Immediately
2 solutions together. Use within 1 day.
before use saturate 120 mL of 6M ammonia with hydrogen
Anunonium Molybdate Solution A 10% w/v solution of su/fide and add 80 mL of 6M ammonia.
ammonium molybdate.
Anunonium Pyrrolidinedithiocarbamate Arnmonium
Ammonium Molybdate Solution R2 Dissolve 5 g of tetramethylenedithiocarbamate; C sH 12N 2S2 = 164.3
ammonium molybdate in 30 mL of water with heating, cool, (5108-96-3)
adjust the pH to 7.0 with 2M ammonia and dilute to 50 mL
with water.
Store in a bottle containing a piece of ammonium carbonate
Ammonium Molybdate Solution R3 in a muslin bago
Solution 1 Dissolve 5 g of ammonium molybdate in 20 mL
Ammonium Pyrrolidinedithiocarbamate Solution A
of water with the aid of heat.
1.0% w/v solution of ammonium pyrrolidinedithiocarbamate
Solution JI Mix 150 mL of ethanol (96%) with 150 mL of that has been washed immediately before use with three
water and add, with cooling, 100 mL of sulfuric acid. 25-mL quantities of 4-methylpentan-2-one.
Add 80 volumes of solution II to 20 volumes of solution 1 Anunonium Reineckate Ammonium
irnmediately before use. tetrathiocyanatodiamminochromate(m) monohydrate;
Anunonium Molybdate Solution R4 Dissolve 1.0 g of NH4[Cr(NH 3 MCNS)4],H20 = 354.4 (13573-16-5)
ammonium molybdate in sufficient water to produce 40 mL, General reagent grade of commerce.
add 3 mL of hydrochloric acid and 5 mL of perchloric acid and Red powder or crystals.
dilute to 100 mL with acetone.
Ammonium Reineckate Solution A 1.0% w/v solution
Store protected from light and use within 1 month of of ammonium reineckate.
preparation.
Prepare irnmediately before use.
Ammonium Molybdate Solution R5 Dissolve 1 g of
ammonium molybdate in 40 mL of a 15% w/v solution of Ammonium Sulfamate Ammonium sulphamate;
sulfuric acid. NH zS03 NH 4 = 114.1 (7773-06-0)
Prepare the solution daily. General reagent grade of commerce.
Ammonium Molybdate Solution R6 Slowly add 10 mL A white, crystalline powder or colourless crystals; melting
of sulfun'c acid R to about 40 mL of water R . Mix and allow to point, about 130°.
cool. Dilute to 100 mL with water R and mix. Add 2.5 g of Store in an airtight container.
ammonium molybdate R and 1 g of cerium sulfate R, and shake Ammonium Sulfate Ammonium sulphate;
for 15 min to dissolve. (NH 4hS04 = 132 .1 (7783-20-2)
Ammonium Molybdate-Sulfuric Acid Solution Analytical reagent grade of commerce.
Arnmonium molybdate-sulfuric acid solution Dissolve 10 g Anunonium Sulfide Solution Ammonium sulphide
of ammonium molybdate in sufficient water to produce solution Saturate 120 mL of dilute ammonia R1 with hydrogen
100 mL and add the solution slowly to 250 mL of cold su/fide and add 80 mL of dilute ammonia R1.
10M sulfuric acid.
Store in a plastic bottle protected from light.
Ammonium Thiocyanate NH 4SCN = 76.12 (1762-95-4)
Ammonium Nitrate NH4N0 3 = 80.04 (6484-52-2)
Store in airtight container.
Ammonium Thiocyanate Solution A 7.6% w/v solution
Ammonium Nitrate Rl Ammonium nitrate complying of ammonium thiocyanate.
with the following tests.
Ammonium Vanadate See Ammonium metavanadate
Acidity A solution is faintly acid, Appendix V K.
Ammonium Vanadate Solution Dissolve 1.2 g of Aniline Hydrochloride Solution Dissolve 2 g of aniline
ammonium metavanadate in 95 mL of water and dilute to hydrochloride in a mixture of 65 mL of ethanol (96%) and
100 mL with sulfuric acid. 35 mL of water and add 2 mL of hydrochloric acid.
Amoxicillin Trihydrate Amoxycillin trihydrate; Use within one day of preparation.
C16H19N30 SS,3H20 = 419.4 (6 1336-70-7) Anion Exchange Resin A resin in chlorinated form
General reagent grade of commerce. containing quatemary ammonium groups [CH 2N +(CH 3h]
Amyl Acetate C 7H 14 0 2 = 130.2 attached ro a polymer latrice consisting of polystyrene cross-
linked with 2% of divinylbenzene. It is available as spherical
Consists principally of 3-methylbutyl acetate with a small
proportion of 2-methylbutyl aceta te. beads and the particle size is specified in the monograph.
Wash the resin with 1M sodium hydroxide on a sintered-glass
Analyrical reagent grade of commerce. filter until the washings are free from chloride, then wash
A colourless liquid; boiling point, about 140°; weight with water until the washings are neutral. Suspend in freshly
per mL, about 0.87 g. prepared ammonia-free water and protect from atmospheric
Amyl Alcohol See lsoamyl alcohol. carbon dioxide .
ex-Amylase l,4-a:-D-glucane-glucanohydrolase Anion Exchange Resin, Strongly Basic A gel-type resin
A white to light brown powder. in hydroxide forrn containing quatemary ammonium groups
[-CH 2 N+(CH 3)3, type 1] attached to a polymer lattice
ex-Amylase Solution A solution of rJ.-amylase with
consisting of polystyrene cross-linked with 8% of
anactivity of 800 FAU per g.
divinylbenzene.
p-Amyrin Olean-12-en-3p-ol;C 30 H so O = 426 .7
Brown, transparent beads containing about 50% of water;
(559-70-6)
particle size, 0.2 mm to 1.0 mm; total exchange capacity, at
White or almost white powder. least 1.2 milliequivalents per mL.
Melting point, 187° to 190°. Anion Exchange Resin for Chromatography, Strongly
Anethole 1-Methoxy-4-(propen-1-yl)benzene; Basic A resin with quatemary amine groups attached to a
C lOH 12 0 = 148.2 (4180-23-8) lattice of latex cross-linked with divinylbenzene.
General reagent grade of commerce. Anion Exchange Resin Rl A resin containing quatemary
A white crystalline mass at 20° to 21°; liquid aboye 23°; ammonium groups [CH 2N+cCH 3)3] attached to a latrice
boiling point, about 230°; n;;, about 1.56. Practically consisting of methacrylate.
insoluble in water, freely soluble in ethanol, soluble in ethyl Anion Exchange Resin R2 A conjugate of homogeneous
acetate and in light petroleum. 10 ¡.1m hydrophilic polyether parricles, and a quatemary
Anethole used in gas chromatography complies with the following ammonium salt, providing a matrix suirable for strong anion-
test. exchange chromatography of proteins.
Assay Examine by gas chromatography, Appendix III B, Anion exchange resin R3 Resin with quatemary
under the conditions described in the test for arnmonium groups attached to a lattice of ethylvinylbenzene
Chromatographic profile in the monograph for Anise Oil crosslinked with 55% of divinylbenzene.
using the reagent being examined as solution (1). Anion Exchange Resin, Weak A resin with
The area of the principal peak, corresponding to diethylaminoethyl groups attached to a lattice consisting of
trans-anethole, with retention time of about 41 minutes, is poly(methyl methacrylate).
not less than 99.0% of the total area of the peaks. Anisaldehyde 4-Methoxybenzaldehyde; C 8 H 8 0 2 = 136.2
ctS- Anethole (Z) -1-Methoxy-4-prop-1-enylbenzene; (123-11-5)
C lOH 12 0 = 148.2 General reagent grade of commerce.
General reagent grade of commerce. A colourless to pale yellow, oily liquid; boiling point, about
A white crystalline mas s at 20°; liquid aboye 23°; boiling 248°; weight per mL, about 1.125 g.
point, about 230°; n;;, about 1.56. Anisaldehyde used in gas chromatography complies with the
Cis-anethole used in gas chromatography complies with the following test. .
following test. Assay Examine by gas chromatography, Appendix III B,
Assay Examine by gas chromatography, Appendix III B, under the conditions described in the test for
under the conditions described in the test for Chromatographic profile in the monograph for Anise Oil
Chromatographic profile in the monograph for Anise Oil using the reagent being examined as solution (1).
using the reagent being examined as solution (1) . The area of the principal peak is not less than 99.0% of the
The area of the principal peak is not les s than 92.0% of the total area of the peaks.
total area of rpe peaks. Anisaldehyde Solution Mix in the following order
Aniline Benzeneamine; C6H7N = 93.1 (62-53-3) 0.5 mL of anisaldehyde, 10 mL of glacial acetic acid, 85 mL
Analytical reagent grade of commerce. of methanol and 5 mL of sulfuric acid.
A colourless or slightly yellowish liquid, soluble in water, Anisaldehyde Solution Rl To 10 mL of anisaldehyde add
miscible with alcohol; boiling point, about 183° to 186°; dig, 90 mL of ethanol (96%), mix, add 10 mL of sulfuric acid and
about 1.02. mix again.
Store protected from light. Anise Ketone 1-(4-Methoxyphenyl)propan-2-one;
Aniline Hydrochloride Benzenamine hydrochloride; C lOH 120 2 = 164.2. (122-84-9)
C 6H 7N,HCl = 129.6 (142-04-1) p-Anisidine C 7H 9NO = 123.2 (104-94-9)
General reagent grade of commerce. General reagent grade of commerce containing not less than
Crystals. It darkens on exposure to air and light, melting 97 .0% of C 7H 9NO.
point, about 198°.
Caution p-Anisidine is a skin imtam and sensitiser. Solution B In a fume cupboard, mix 100 roL of
Store protected from light at 0° to 4°. colourless, distilled acetyl chloride and 400 mL of
On storage, p-anisidine tends to darken as a result of
l,2-dichloroethane and store in a cool place.
oxidation. A discoloured reagent can be reduced and Mix 90 mL of solution A and 10 mL of solution B. Store in
decolorised in the following manner. Dissolve 20 g of the an amber, ground-glass-stoppered bottle and use within
reagent in 500 mL of water at 75°. Add 1 g of sodium sulfite 7 days. Discard any reagent in which colour develops.
and 10 g of activated eharcoal and stir for 5 minutes. Filter, Antithrombin III ATIII (90170-80-2)
cool the filtrate to about 0° and allow to stand at this Reagent grade of commerce.
teroperature for at least 4 hours. Filter, wash the crystals with
Antithrombin III is purified from human plasma by heparin
a small quantity of water at about 0° and dry the crystals at a agarose chromatography and should have a specific activity of
pressure of 2 kPa over phosphorus pentoxide. at least 6 IU per mg.
Anolyte for Isoelectric Focusing pH 3 to 5 O.lM
Antithrombin In Solution Rl Reconstitute
glutamic acid, 0.5M phosphoric acid. Dissolve 14.7 1 g of antithrombin III as directed by the manufacturer and dilute
glutamie acid in water, add 33 mL of onhophosphon'e acid and with tris-chlon'de buffer pH 7.4 to contain 1 IU per mL.
dilute to 1000 mL with water.
Antithrombin In Solution R2 Reconstitute
Anthracene C I4 H JO = 178.2 (120-12-7)
antithrombin III as directed by the manufacturer and dilute
General reagent grade of commerce. with tris-ehlonde buffer pH 7.4 to contain 0.5 IU per mL.
A wrute, crystalline powder; melting point, about 218°.
Antithrombin In Solution R3 Reconstitute antithrombin
Anthranilic Acid See 2-Aminobenzoie acid. III as directed by the manufacturer and dilute to 0.3 IU/roL
Anthrone C I4 HJOO = 194.2 (90-44-8) with phosphate buffer solution pH 6.5.
Analytical reagent grade of commerce. Antithrombin nI Solution R4 Reconstitute antithrombin
A pale yellow, crystalline powder; melting point, about 155°. III as directed by the manufacturer and dilute to 0.1 IU/roL
When used in an assay for glucose or dextrans, eomplies with the with tris(hydroxymethyl)aminomethane EDTA buffer solution
following test. pH 8.4.
Sensitivity to glucose Add, carefully, 6 mL of a 0.2% w/v Apigenin 4',5,7-Trihydroxyflavone; C1 sH JOO = 270.2
solution in a mixture of 19 mL of sulfurie acid and 1 mL of (520-36-5)
water to 3 roL of a solution containing 5 ¡.¡g of D-glueose
per mL and heat in a water bath for 5 minutes. The solution
is a darker green than a solution prepared in the same Light yellowish powder.
manner but omitting the glucose. Melting point, about 310°, with decomposition.
Anthrone Reagent A 0.2% w/v solution of anthrone in Complies with the following test.
nitrogen-free sulfurie acid. The solution should be allowed to Chromatography Carry out identification test C
stand for 4 hours before use and should be discarded after described under Chamomile Flowers applying 10 ¡.¡L of a
7 days. 0.025 % w/v solution in methanol. The chromatogram shows
Antimony Potassium TartrateAntimony potassium principal zone of yellowish-green fluorescence in the upper
oxide (+)-tartrate; KSbO,C 4H 4 0 6, '/2 H zO = 333.9 thlrd.
(28300-74-5) Apigenin-7-glucoside CZIHzoOJO = 432.6
Analytical reagent grade of commerce. General reagent grade of commerce.
Antimony Trichloride SbCI 3 = 228.1 (10025-91-9)
Light yellowish powder.
Melting point, 198° to 201 0 •
Colourless crystals or flakes, fuming in moist air.
Complies with the following test.
Store in an airtight container, protected from moisture.
Antimony Trichloride in Dichloroethane Solution Chromatography Carry out identification test C
Prepare a 22.0% w/v solution of antimony triehloride in dry described under Chamomile Flowers applying 1O ~lL of a
l,2-diehloroethane that has been purified by passing it down a 0.025% w/v solution in methanol. The chromatogram shows
column of siliea gel and allow the solution to stand for 24 a principal zone of yellowish fluorescence in the middle
hours . To 100 mL add 2.5 mL of aeetyl chloride and allow to trurd.
stand for a further 24 hours before use. Apigenin-7-glucoside used in liquid ehromatography complies with
Antimony Trichloride Solution Rapidly wash 30 g of the following additional test.
amimony trichloride with two 15-roL quantities of ethanol-free Assay Examine by liquid chromatography (2.2.29) as
chloroform. Drain off the washings and dissolve the washed prescribed in the monograph on Matricaria fiower.
crystals irnmediately in 100 roL of ethanol-free chloroform, Test solution Dissolve 10.0 mg in methanol R and dilute to
warming slightly. 100.0 mL with the same solvent.
Store over a few grams of anhydrous sodium sulfate. The content of apigenin-7-glucoside is not less than 95.0%,
Antimony Trichloride Solution Rl Prepare two stock ca1culated by the normalisation procedure .
solutions in the following manner. Apiole Apiol; apioline; parsley apiole; 5-allyl-4,7-
Solution A Dissolve 110 g of antimony trichloride in dimethoxy-1,3-benzodioxol; Cl zHI404 = 222.24 (484-31-1)
400 mL of l ,2-diehloroethane. Add 2 g of anhydrous General reagent grade of commerce.
aluminium oxide, mix and filter through sintered glass into a
Aprotinin (9087-70-1)
500-mL graduated flask. Dilute to 500 mL with
l,2-diehloroethane and mix. The absorbance of the resulting Reagent grade of commerce containing lOto 20 trypsin
solution measured in a 2-cm cell at 500 nm, Appendix II B, inhibitor units per mg.
is not more than 0.07 using l,2-dichloroethane in the Arabinose See L-Arabinose.
reference cel!.
L-Arabinose Arabinose; CSHlOOS = 150.1 (87-72-9) A white, crystalline powder; [a]~, about + 22 (2% w/v in
General reagent grade of commerce. water).
A white, crystalline powder; [a]~o, + 103 to + 105 (5.0% w/v Ascorbic Acid Solution Dissolve 50 mg of L-ascorbic acid
in water containing about 0.05 % v/v ofNH 3 ). in 0.5 mL of water and dilute to 50 mL with
Arachidic Alcohol Eicosan-1-ol; arachidyl alcohol; dimethylfonnamide .
C 2oH 42 0 = 298.6 (629-96-9) Asiaticoside 0-6-Deoxy-IX-L-mannopyranosyl- ( 1 -> 4)-O-~
Purified reagent grade of commerce usually containing not D-glucopyranosyl-(l-> 6)-~-D-glucopyranosyl 21X,3~,23-
less than 95 % of C 2oH 420. trihydroxy-41X-urs-12-en-28-oate;C 48H 7s O 19 = 959
(16830-15-2)
A colourless, waxy or crystalline solid .
General reagent grade of commerce containing not more
Arachidyl Alcohol See Arachidic Alcohol. than 6.0% of water.
Arbutin Arbutoside; 4-hydroxyphenyl-~-D-glucopyranoside; Melting point, about 232°, with decomposition.
Cl2H1607 = 272.3 (497-76-7)
Store protected from humidity.
Asiaticoside used in liquid chromatography complies with the
Melting point, about 200°; [a]~, about - 64, detennined on a folw wing test.
2.0% w/v solution.
Assay When examined by the method described under
Chromatography When examined by test C for Assay in the monograph for Centella, the content is not less
Identification in the monograph for Bearberry Leaf the than 97.0% by nonnalisation.
chromatogram shows onIy one principal spot.
Aspartic Acid (56-84-8)
Arbuuil used in the arbutin assay in the monograph for Bearbeny
Leaf complies with the following additional test. General reagent grade of commerce.
Assay Carry out the method as described in the L-AspartyI-L-phenylalanine Aspartame; (S)-3-amino-
N- [(S)~ l-carboxy-2-phenylethyl] succinamic acid;
monograph for Bearberry Leaf. The content of arbutin is not
les s than 95%, calculated by the nonnalisation procedure. C13HI6N20S = 280.3 (13433-09-5)
Arginine L-Arginine; C6Hl 4N40 2 = 174.2 (74-79-3)
General reagent grade of commerce. Melting point, about 210°, with decomposition.
Melting point, about 235°, with decomposition. Astragaloside IV (20R,24S) -20,24-Epoxy-16~,25-
dihydroxy-3 ~-(~-D-xylopyranosyloxy)-9, 19-cyc1olanostan-
Argon Ar = 39.95 (7440-37-1) 61X-yl ~-D-glucopyranoside; C41H68014 = 785 (84687-43-4)
Laboratory cylinder grade of commerce containing not less Ateno101 C1 4H 22N203 = 266.3 (56715-13-0)
than 99.995 % v/v of Ar.
When used in the test for Carbon monoxide in medicinal
gases (Appendix IX E), after passage of 10 litres at a ftow Atropine Sulfate Atropine sulphate;
rate of 4 litres per hour, not more than 0.05 mL of C34H46N20 6,H2S04,H20 = 694 .8 (5908-99-6)
0.002M sodium thiosulfate VS is required for the titration Of the British Pharmacopoeia.
(0.6 ppm) . Aucubin (1S,4aR,5S, 7aS)-5-Hydroxy-7 -(hydroxyrnethyl)-
Argon Rl Ar = 39.95 (7440-37-1) 1,4a,5, 7a-tetrahydrocyc1openta[c)pyran-l-yl ~-D
Content, minimum 99.99990% v/v. glucopyranoside; C 1s H 22 0 9 = 346 .3 (479-98-1)
Argon for Chromatography Ar = 39.95 (7440-37-1) Crystals, soluble in water, in alcohol and in methanol,
practically insoluble in light petroleum.
Analytical grade of commerce. The content is ininimum
[a]~o: about - 163. Meltingpoint, about 181 °.
99 .95% VN of Ar.
Aromadendrene (lR,2S,4R,8R,11R)-3,3,11 -Trimethyl- Azadirachtin C3sH4401 6 = 720.7 (11141-17-6)
7-methylenetricyc1o [6.3.0.0 2,4] undecane General reagent grade of commerce.
General reagent grade of commerce. Melting point, about 165°.
d~o, about 0.9ll; n~o, 1.497; [a]~, about 12; boiling point, Azobenzene C l2H lON 2 = 182.2 (103-33-3)
about 263°. Use a grade of commerce suitable for chromatographic
Aromadendrene used in gas chromatography complies with the standardisations.
following test. Melting point, about 69°.
Assay Carry out the method for chromatographic profile Azomethine H Azomethine H , monosodium salt hydrate;
described in the monograph for Tea Tree Oil. The content sodium hydrogen 4-hydroxy-5-
is not less than 92%, calculated by the normalisation (2-hydroxybenzylideneamino) na phthalene-2, 7-disulfonate;
procedure. C17HI2NNaOSS2 = 445.4 (5941 -07-1)
Arsenious Trioxide Arsenic trioxide; AS 20 3 "7 197.8 General reagent grade of commerce; the degree of hydration
(132 7-53-3) is variable.
Analytical reagent grade of commerce. Azomethine H SoIution. Dissolve 0.45 g Df azomethine H
Arsenite Solution Dissolve 0.50 g of arsenious trioxide in and 1 g of ascorbic acid in water with the aid of gentle heat
5 mL of 2M sodium hydroxide solution, add 2.0 g of sodium and add sufficient water to produce 100 mL.
hy drogen carbonate and dilute to 100 mL with water. Baicalin 5,6-Dihydroxy-4-oxo-2-phenyl-4H-1-benzopyran-
Ascorbic Acid See L-Ascorbic acid. 7 -yl-~-D-glucopyranosiduronic acid;
L-Ascorbic Acid Ascorbic acid; C 6H s0 6 = 176.1 C21H1SOll = 446.4 (21967-41-9)
(50-81-7)
Barbaloin Aloin; 1,8-Dihydroxy-3-hydroxyrnethyl- White or pale yellow mass.

1O-(~-D-glucopyranosyl)anthrone; Content, minimum 98.0%; boiling point, about 261 °;
C21H 220 9,H20 = 436.4 (1415-73-2) melting point, about 39°.
Yellow to dark-yellow, crystalline powder, or yellow needles, Benzaldehyde C 7H 60 = 106.1 (100-52-7)
darkening on exposure to air and light, sparingly soluble in Colourless or slightly yellow liquid, slightly soluble in water,
water and in ethanol (96%), soluble in acetone, in ammonia miscible with ethanol (96%).
and in solutions of alkali hydroxides.
A(l %, 1 cm) about 192 at 269 nm, about 226 at 296.5 nm,
dig, about 1.05; n~o, about 1.545.
about 259 at 354 nm, determined on a solution in methanol Distillation range Not less than 95% distils between 17T
and calculated with reference to the anhydrous substance. and 180°.
Complies with the follo wing test.
Homogeneity Carry out the test for Other species of Benza1konium Chloride Of the British Pharmacopoeia.
Rhamnus, anthrones described under Frangula bark applying Benzalkonium Chloride Solution Of the British
to the plate a solution containing only the reagent being Pharmacopoeia.
examined. The chromatogram obtained shows only one Benzene C6H6 = 78.1 (71-43-2)
principal spot. Clear, colourless, fiammable liquid, practically insoluble in
Barbital Of the British Pharmacopoeia. water, miscible with ethanol (96%).
Barbital Sodium Sodium derivative of 5,5-diethyl- Boiling point, about 80 0 •
lH,3H,5H-pyrimidine-2,4,6-trione; Benzene-l,2,4-triol Hydroxyhydroquinone,
CSHllN2Na03 = 206.2 (144-02-5) hydroxyquinol; C 6H 60 3 = 126.1 (533-73-3)
Content, minimum 98.0%. Freely soluble in water, in ethanol (96%) and in ethyl
A white or almost white, crystalline powder or colourless acetate.
crystals, freely soluble in water, slightly soluble in ethanol Melting point, about 140°.
(96%). Benzethonium Chloride Benzyldimethyl [2-[2- [4-(1 , 1,3,3-
Barbitone See Barbital tetramethylbutyl)phenoxyJethoxyJethylJarnmonium chloride
Barbitone Sodium See Barbital sodium monohydrate; C27H42CIN02,H20 = 466.1 (121-54-0)
Barbituric Acid 2,4,6-Trihydroxypyrimidine; Fine, white or almost white powder or colourless crystals,
C4H 4N 20 3 = 128.1 (67-52-7) soluble in water and in ethanol (96%).
White or almost white powder, slightly soluble in water, Melting point, about 163 0 •
freely soluble in boiling water and in dilute acids. Store protected from light.
Melting point, about 253°. Benzidine Biphenyl-4,4 '-diamine;
Barium Acetate Barium di aceta te; C 12H 12 N 2 = 184.2 (92-87-5)
C4H6Ba04 = 255.4 (543-80-6) Content, minimum 95%.
White or almost white powder, soluble in water. White or slightly yellowish or reddish powder, darkening on
dig,2.47. exposure to air and light.
Barium Carbonate BaC0 3 = 197.3 (513-77-9) Melting point, about 120 0
•
White or almost white powder or friable mas ses, practically Store protected from light.
insoluble in water. Benzil Diphenylethanedione;
Barium Chloride Barium dichloride; C 14HlQ02 = 210.2 (134-81-6)
BaClz,2H20 = 244.3 (10326-27-9) Yellow, crystalline powder, practically insoluble in water,
Colourless crystals, freely soluble in water, slightly soluble in soluble in ethanol (96%), ethyl acetate and toluene.
ethanol (96%). Melting point, 95°.
Barium Chloride Solution A 10.0% w/v solution of Benzocaine Of the British Pharmacopoeia.
barium ehloride.
Benzoic Acid Of the British Pharmacopoeia.
Barium Chloride Solution Rl A 6.1 % w/v solution of Benzoin rJ.- Hydroxy-a-phenylacetophenone;
barium ehloride.
C14H1 20 2 = 212 .3 (579-44-2)
Barium Chloride Solution R2 A 3.65% w/v solution of Slightly yellowish crystals, very slightly soluble in water, freely
barium ehloride.
soluble in acetone, soluble in hot ethanol (96%).
Barium Hydroxide Barium dihydroxide; Melting point, about 137°.
Ba(OH)z,8H 20 = 315.5 (12230-71-6)
Benzophenone Diphenylmethanone;
Colourless crystals, soluble in water C 13 H lO O = 182.2 (119-61-9)
Barium Hydroxide Solution A 4.73 % w/v solution of Prismatic crystals, practically insoluble in water, freely soluble
barium hydroxide.
in ethanol (96%).
Barium Nitrate Ba(N03)z = 261.3; (10022-31-8) Melting point, about 48°.
Crystals or crystalline powder, freely soluble in water, very 1,4-Benzoquinone Cyclohexa-2,5-diene-l,4-dione;
slightly soluble in ethanol (96%) and in acetone; melting C 6H 40 2 = 108.1 (106-51-4)
point, about 590°.
Content, minimum 98.0%.
Barium Sulfate Of the British Pharmacopoeia.
Benzoylarginine Ethyl Ester Hydrochloride N-Benzoyl-
Benzalacetone (3E)-4-phenylbut-3-en-2-one; L-arginine ethyl ester hydrochloride, ethyl (S)-2-benzamido-
ClQHlQO = 146.2 (122-57-6) 5-guanidinovalerate hydrochloride;
CI 5H23CIN40 3 = 342.8 (2645-08-1) YeIlow liquido

White or almost white, crystaIline powder, very soluble in Melting point, 13° ro 16°.
water and in anhydrous ethano!. 4-Benzy1pyridine C12HllN = 169.2 (2116-65-6)
[a]g>, -15 ro - 18, determined in a 1% w/v solution. Content, minimum 98.0%.
Melting point, about 129°. YeIlow liquido
A( 1%, 1 cm) , 310 to 340 determined at 227 nm using a Melting point, 72° ro 78°.
0.001 % w/v solution. Benzy1trimethy1ammonium Chloride
Benzoy1 Chloride C 7 H 5CIO = 140.6 (98-88-4) Trimethylphenylmethanaminium chloride;
ColourIess, lachrymarory liquid, decomposed by water and C IO H 16 CIN = 185.7 (56-93-9)
by ethanol (96%). White or almost white powder, soluble in water.
d~g, about 1.21; boiling point, about 197
0
•
Melting point, about 230°, with decomposition.
Benzoy1ecgonine Hydrate Berberine Chloride 9,10-Dimethoxy-5,6-
C16H19N04,H20 = 307.3 (519-09-5) dihydro benzo [g]-1 ,3-benzodioxolo [5, 6-a] quinolizinium
General reagent grade of commerce. chloride; C2oH1 SCIN04,2H20 = 407 .8 (5956-60-5)
Benzoy1 Peroxide C 14H IO 0 4 = 242.2 (94-36-0) YeIlow crystals, slightly soluble in water, practicaIly insoluble
General reagent grade of commerce. in ethanol (96%).
White or almost white granules; melting point, after drying, Melting point, 204° to 206°.
about 104°. Berberine chlande used in liquid chramatography camplies with the
For safety Benzoyl Peroxide should be kept moistened with fallowing test.
about 23% w/w of water. Assay Examine by liquid chromatography (2.2.29) as
N- Benzoy1-L-pro1y1-L-pheny1alany1-L-arginine prescribed in the monograph on Goldenseal root (1831).
4-Nitroanilide Acetate C35H42NsOs = 703 The content is not less than 95%, calculated by the
General reagent grade of commerce. narmalisatian procedure.
3-Benzoy1propionic Acid 4-0xo-4-phenylbutanoic acid; Bergapten 5-Methoxypsoralen; C 12H S 0 4 = 216 .2
(484-20-8)
C IOH IO 0 3 = 178.2 (2051-95-8)
Melting point, about 118°. ColourIess crystals, practicaIly insoluble in water, sparingly
soluble in ethanol (96%) and slightly soluble in glacial acetic
2-Benzoy1pyridine Benzoylpyridine, Phenyl(pyridin- acid.
2-yl)methanone; C 12H 9 NO = 183.2 (91-02-1)
Colour1ess crystals, soluble in ethanol (96 %).
Betamethasone 9 <x-Fluoro-ll~, 17<x,21-trihydroxy-
Melting point, about 43°. 16~-methylpregna-1,4-diene- 3,20-dione;
Benzy1 Alcohol Of the British Pharmacopoeia. C 22 H 29 F0 5 = 392.5 (378-44-9)
Benzy1 Benzoate Of the British Pharmacopoeia. General reagent grade of commerce.
Benzy1 Cinnamate Benzyl 3-phenylprop-2-enoate; Melting point, about 236°.
C 16H 14 0 2 = 238.3 (103-41-3) Betulin Lup-20(39)-ene-3~,28-diol;
ColourIess or yeIlowish crystals, practicaIly insoluble in water, C30H5002 = 442.7 (473-98-3)
soluble in ethanol (96%).
White or almost white, crystaIline powder.
Melting point, about 39°. Melting point, 248° to 251 0 .
Camplies with the fallawing test.
Bibenzy1 1,2-Diphenylethane; C 14H 14 = 182 .3 (103-29-7)
Chromatography Carry out test B for Identification White or almost white, crystaIline powder, practicaIly
described in the monograph for Peru Balsam applying to the insoluble in water, very soluble in methylene chloride, freely
plate 20 ).lL of a 0.3 % w/v solution of the reagent being soluble in acerone, soluble in ethanol (96%).
examined in ethyl acetate. After visualisation, the
chromatogram exhibits a principal spot with an Rf value of Melting point, 50° to 53°.
about 0.6 . Bipheny1 C 12H lO = 154.2 (92-52-4)
Benzy1 Cyanide Phenylacetonitrile; Melting point, 68° to 70°.
C SH 7 N = 11 7.2 (140-29-4) Biphenyl-4-o1 4-Phenylphenol;
Content, minimum 95.0%. C l2H lOO = 170.2 (90-43-7)
Clear, colourIess or light yeIlow liquid. White or almost white, crystaIline powder, practicaIly
ng>, about 1.523; boiling point, about 233°. insoluble in water.
Benzy1 Ether Dibenzyl Ether; Melting point, 164° to 167°.
C 14H 140 = 198.3 (103-50-4) (-)-<x-Bisabo101 (2S)-6-Methyl-2-[(1S)-4-methylcyc1ohex-
Clear, colourIess liquid, practicaIly insoluble in water, 3-enyl]hept-5-en-2-ol, Levomenol;
miscible with acetone and with anhydrous ethano!. C 15H 26 0 = 222.4 (23089-26-1)
dig, about 1.043; n~o, about 1.562; boiling point, about ColourIess, viscous liquid with a slight, characteristic odour,
296°, with decomposition. practicaIly insoluble in water, freely soluble in ethanol (96%),
in methanol, in toluene, in fatty oils and in essential oils.
Benzy1penicillin Sodium Of the British Pharmacopoeia.
2-Benzy1pyridine C l2H ll N = 169.2 (101-82-6)
dig, 0.925 ro 0.935; ng>, 1.492 to 1.500; [a]~o, - 54.5 to
- 58.0, determined on a 5% w/v solution in ethanol (96%).
(-) -rx-Bisabalol used far gas chramatography camplíes with the
fallawing additianal test.
Assay Gas chromatography (2.2.28) as prescribed in the Add 300 mL of a carbonate-free 10% w/v solution of sodium
monograph Matricaria oil (1836) . hydroxide and sufficient of the same solution to produce
Test solution A 0.4% w/v solution in cyclohexane. 1000 mL and mix.
Content, mirtimum 95.0%, caJculated by the normalisation Blocking Solution A 10% v/v solution of acetic acid.
pro ce dure. Blue Dextran 2000 (9049-32-5) Prepared from dextran
Bisbenzimide 4- {5- [5-( 4-Methylpiperazin- having an average molecular weight of about 2 x 10 6 by
1-yl)benzimidazol-2-yl] benzimidazol-2-yl}phenol introduction of a polycyclic chromophore that colours the
trihydrochloride pentahydrate; substance blue . The degree of substitution is 0.017. It is
CZSH24N60,3HCI,5H20 = 624 (23491-44-3) freeze-dried and dissolves rapidly and completely in water
General reagent grade of commerce. and aqueous saline solutions.
Bisbenzimide Stock Solution Dissolve 5 mg of Complies with the following test.
bisbenzimide in sufficient water to produce 100 mL. Light absorption A 0.1 % w/v solution in phosphate buffer
Store protected from light. pH 7.0 exhibits a maximum at 280 nm, Appendix TI B.
Bisbenzimide Working Solution Irnrnediately before use, Boldine Cl9H21N04 = 327.3 (476-70-0)
dilute 100 ¡.tL of bisbenzimide stock solution to 100 mL with White or almost white crystalline powder, very slightly
phosphate-buffered saline pH 7.4. soluble in water, soluble in ethanol (96%) and in dilute
Bismuth Nitrate Pentahydrate solutions of acids.
Bi(N0 3h,5H20 = 485.1 (10035-06-0) [a]b", about + 127 determined in a 0.1 % w/v solution in
Me1ting point, about 30°. absolute ethanol.
Bismuth Oxycarbonate Bismuth subcarbonate Melting point, about 163 0
•
(5892-10-4) Borate Solution Dissolve 9.55 g of sodium tetraborate in

A basic salt corresponding approximately to the formula sulfuric acid (96% w/w), heating on a water bath, and add
(BiO)zC0 3 y2H zO, containing the equivalent of about 91 % sufficient sulfuric acid (96% w/w) to produce 1000 mL.
ofBi 20 3 · Boric Acid H 3 B03 = 61.83 (10043-35-3)
Bismuth Oxynitrate See Bismuth subnitrate Analytical reagent grade of commerce.
Bismuth Oxynitrate Rl See Bismuth subnitrate Rl Borie Acid Solution Dissolve 5 g of bon'c acid in a
Bismuth Oxynitrate Solution See Bismuth subnitrate mixture of 20 mL of water and 20 mL of absolute ethanol and
solution dilute to 250 mL with absolute ethanol.
Bismuth Subcarbonate See Bismuth oxycarbonate. Borie Acid SoIution, Cold Saturated To 3 g of boric
acid add 50 mL of water and shake for 10 minutes. Place the
Bismuth Subnitrate Bismuth oxynitrate; solution for 2 hours in a refrigerator.
4[BiN0 3 (OH 2),BiO(OH)] = 1462 (1304-85-4)
Borneol D-Borneol, endo- l, 7,7-
White or almost white powder, practically insoluble in water. trimethylbicyclo[2.2.1]heptan-2-01;
Bismuth Subnitrate Rl CjQH1 SO = 154.3 (507-70-0)
Content, 71.5% to 74.0% ofbismuth (Bi), and 14.5% to Colourless crystals, readily sublimes, practically insoluble in
16.5% ofnitrate, caJculated as nitrogen pentoxide (N20 S)' water, freely soluble in ethanol (96%) and in light petroleum.
Bismuth Subnitrate Solution Bismuth oxynitrate Melting point, about 208°.
solution.
Homogeneity Carry out the method for thin-layer
Dissolve 5 g of bismuth oxynitrate R1 in a mixture of 8.4 mL chromatography, Appendix 111 A, using silica gel G as the
of nitric acid and 50 mL of water and dilute to 250 mL with coating substance and chloroform as the mobile phase, but
water. Filter if necessary. allowing the solvent front to ascend 10 cm aboye the line of
Acidity To 10 mL of the reagent add 0.05 mL of methyl application. Apply to the plate 10 ¡.tL of a 0.1 % w/v solution
orange solution and titrate with 1M sodium hydroxide VS. Not of the substance being examined in toluene. After removal of
les s than 5.0 mL and not more than 6.25 mL is required to the plate, allow it to dry in air and spray with anisaldehyde
change the colour of the solution. solution, using 10 mL for a plate 200 mm x 200 mm, and
N, 0-Bis(trimethylsilyl)acetamide heat at 100° to 105° for 10 minutes. The chromatogram
C SH 21NOSi 2 = 203.4 (10416-59-8) obtained shows only one principal spot.
General reagent grade of commerce. n-Borneol See Borneol
A colourless liquid; weight per mL, about 0.83 g. BornyI Aeetate D-Bornyl Acetate;
Bis (trimethylsilyl)trifluoroacetamide BSTFA; Cl2H200 2 = 196.3 (5655-61-8)
CSHlSF3NOSi2 = 257.4 (25561-30-2) Colourless crystals or a colourless liquid, very slightly soluble
General reagent grade of commerce. in water, soluble in ethanol (96%).
Weight per mL, about 0.97 g; ni,°, about 1.384; boiling Melting point, about 28°.
point, about 40°. Complies with the following test.
Biuret C 2H sN 30 2 = 103 .1 (108-19-0) Homogeneity Carry out the method for thin-layer
White or almost white crystals, hygroscopic, soluble in water, chromatography, Appendix III A, using silica gel G as the
sparingly soluble in ethanol (96%). coating substance and chloroform as the mobile phase, but
0
allowing the solvent front to ascend 10 cm aboye the line of
Melting point, 188° to 190 with decomposition.
,
application. Apply to the plate 10 ¡.tL of a 0.2% w/v solution
Store in an airtight container. of the reagent being examined in to/uene. After removal of
Biuret Reagent Dissolve 1.5 g of copper(lI) sulfate and the plate, allow it to dry in air and spray with anisaldehyde
6.0 g of potassium sodium (+)-tartrate in 500 mL of water. solution, using 10 mL for a plate 200 mm x 200 mm, and
heat at 100° to 105° for 10 minutes. The chromatogram Bromine Water A saturated solution obtained by shaking
obtained shows only one principal spot. occasionaIly during 24 hours 3 mL of bromine with 100 mL
o-Bornyl Acetate See Bornyl acetate. of water and aIlowing to separate.
Boron TrichIoride BCI3 = 117.2 (10294-34-5) Store the solution over an excess of bromine, protected from
light.
ColourIess gas. Reacts violentIy with water. Available as
solutions in suitable solvents (2-chloroethanol, Bromine Water Rl Shake 0.5 mL of bromine with
dichloromethane, hexane, heptane, methanol). 100 mL of water.
Boiling point, about 12.6°; n~o, about 1.420. Store protected from light, use within 1 week.
Cauuon: toxic and corrosive. Bromobenzene C6H5Br = 157.0 (108-86-1)
Boron TrichIoride-Methanol Solution A 12% w/v General reagent grade of commerce.
solution of BCI 3 in methanol. A colourIess to pale yeIlow liquid; boiling point, about 156°;
Store protected from light at -20°, preferably in sealed tubes. n~o, about 1.56; weight per mL, about 1.50 g.
Boron Trifluoride BF3 = 67.8 (7637-07-2) Bromocresol Green 4,4 '-(3H-2, I-Benzoxathiol-
3-ylidene)bis(2,6-dibromo-m-cresol) S,S-dioxide;
Colourless gas.
C21H14Br405S = 698 (76-60-8)
Boron Trifluoride-Methanol Solution A 14% w/v
solution of boron trifiuoride in methanol. Brownish-white powder, slightIy soluble in water, soluble in
ethanol (96%) and in dilute solutions of alkali hydroxides.
Boron Trifluoride Solution See Boron trifiuoride-methanol
Bromocresol Green-Methyl Red Solution Dissolve
solution
0.15 g of bromocresol green and 0.1 g of methyl red in 180 mL
Bovine Coagulation Factor Xa An enzyme which of absolute ethanol and dilute to 200 mL with water.
converts prothrombin to thrombin. The semi-purified
preparation is obtained from liquid bovine plasma and it may Bromocresol Green Solution Dissolve 50 mg of
be prepared by activation of the zymogen factor X with a bromocresol green in 0.72 mL of 0.1 Msodium hydroxide and
suitable activator such as RusseIl's viper venom. 20 mL of ethanol (96%). After solution is effected add
Store freeze-dried preparation at -20° and frozen solution at
a temperature not higher than -20°. Complies with the following tests.
Sensitivity A mixture of 0.2 mL of the solution and
Bovine Factor Xa Solution Reconstitute bovine
coagulation factor Xa as directed by the manufacturer and 100 mL of carbon dioxide-free water is blue. Not more than
dilute with tris-chloride buffer pH 7.4. Any change in the 0.2 mL of 0.02M hydrochloric acid VS is required to change
absorbance of the solution, Appendix JI B, measured at the colour of the solution to yelIow.
405 nm using tris-chloride buffer pH 7.4 in the reference ceIl, Colour change pH 3.6 (yeIlow) to pH 5.2 (blue) .
is not more than 0.15 to 0.20 per minute. Bromocresol Purple 4,4'-(3H-2,I-Benzoxathiol-
Bovine Factor Xa Solution Rl Reconstitute as directed 3-ylidene) bis (6-bromo-o-cresol) S,S-dioxide;
by the manufacturer and dilute to 1.4 nkatlmL with C21Hl6Br205S = 540.2 (115-40-2)
tris(hydroxymethyl)aminomethane EDTA buffer solution pH 8.4. Pinkish powder, practicalIy insoluble in water, soluble in
Brilliant Blue See Acid blue 83. ethanol (96%) and in dilute solutions of alkali hydroxides.
Brilliant Green CI 42040; basic green 1; Bromocresol Purple Solution Dissolve 50 mg of
C27H34N204S = 482.6 (633-03-4) bromocresolpurple in 0.92 mL ofO.lMsodium hydroxide and
Technical grade of cornmerce. 20 mL of ethanol (96%) and add sufficient water ro produce
SmaIl, glistening golden crystals. 100 mL.
Bromelains (37189-34-7) Concentrate of proteolytic Complies with the following tests.
enzymes derived from Ananas comosus Merr. Sensitivity A mixture of 0.2 mL of the solution and
DuIl-yeIlow powder. 100 mL of carbon dioxide-free water ro which 0.05 mL of
Complies with the following test. 0.02M sodium hydroxide VS has been added is bluish violet.
Activity 1 g liberates about 1.2 g of amino nitrogen from a
Not more than 0.2 mL of 0.02M hydrochloric acid VS is
required to change the colour of the solution to yelIow.
standard gelatin solution in 20 minutes at 45° and pH 4.5.
Bromelains Solution A 1.0% w/v solution of bromelains in Colour change pH 5.2 (yeIlow) to pH 6.8 (bluish-violet).
a mixture of 1 volume of mixed phosphate buffer pH 5.5 and 5-Bromo-2' -deoxyuridine
9 volumes of saline soluuon. C 9H Il BrN20 5 = 307.1 (59-14-3)
Bromine Br2 = 159.8 (7726-95-6) Melting point, about 194°.
Brownish-red fuming liquid, slightIy soluble in water, soluble Complies with the following test.
in ethanol (96%). Homogeneity Examine under the conditions prescribed in
dig, about 3.1. the test for Related substances in the monograph for
To prepare 0.05M bromine dissolve 3 g of potassium bromate ldoxuridine applying to the plate 5 ¡.tL of a 0.025% w/v
and 5 g of potassium bromide in sufficient water to produce solution. The chromatogram shows onIy one principal spot.
1000 mL. Weaker solutions should be prepared using Bromomethoxynaphthalene 2-Bromo-
proportionately lesser amounts of reagents or by appropriate 6-methoxynaphthalene; CIlH9BrO = 237.1 (5111-65-9)
dilution.
Bromine Solution Dissolve 30 g of bromine and 30 g of
Bromophenol Blue 4,4'-(3H-2, l-Benzoxathiol-3-ylidene)
potassium bromide in sufficient water to produce 100 mL.
bis(2,6-dibromophenol) S,S-dioxide;
Bromine Solution, Acetic Dissolve 100 g of potassium
C19HIOBr405S = 670 (115-39-9)
acetace in glacial acetic acid and add 4 mL of bromine and
sufficient glacial acetic acid to produce 1000 mL.
Light orange-yeIlow powder, very slightIy soluble in water, ButanaI Butyraldehyde; C 4H sO = 72.1 (123-72-8)
slightly soluble in ethanol (96%), freely soluble in solutions d~g, 0.806; n~o: 1.380; boiling point, 75°.
of alkali hydroxides. Butane-l,3-dioI C4H\OOz = 90.1 (6290-03-5)
BromophenoI BIue SoIution Dissolve 0.1 g of General reagent grade of commerce.
bromophenol blue in 1.5 mL of O.lM sodium hydroxide and
20 mL of ethanol (96%) and add sufficient water to produce Boiling point, about 203°; n~, about 1.440.
100 mL. Butane-l,4-dioI HO(CHz)40H = 90.12 (110-63-4)
COl11plies with the following tests. General reagent grade of commerce.
Sensitivity A mixture of 0.05 mL of the solution and ButanoI Butan-1-ol; 1-butanol; n-butanol; n-Butyl alcohol;
20 mL of carbon dioxide-free water to which 0.05 mL of O.lM C 4 H\OO = 74.1 (71-36-3)
hydrochloric acid VS has been added is yellow. Not more than Clear, colourless liquid, miscible with ethanol (96%).
0.1 mL of O.lM sodium hydroxide VS is required to change d~g, about 0.81; boiling point, 116 to 119
0 0
the colour to bluish violeto Butan-l-oI See Butanol

Colour change pH 2.8 (yellow) to pH 4.4 (bluish-violet). 2-Butanol Rl sec-Butyl alcohol; C 4H IO O = 74.1 (78-92-2)
BromophenoI BIue SoIution Rl Dissolve 50 mg of Content, minimum 99.0%.
bromophenol blue with gentle heating in 3.73 mL of 0.02M
sodium hydroxide and dilute to 100 mL with water. Clear, colourless liquid, soluble in water, miscible with
ethanol (96%).
Bromophenol BIue SoIution R2 Dissolve with heating
0.2 g of bromophenol blue in 3 mL of O.lM sodium hydroxide d~g, about 0.81.
and 10 mL of ethanol (96%) . Allow to cool and dilute to Distillation range Not less than 95% distils between 99°
100 mL with ethanol (96%) . and IOO°, Appendix V C.
Bromophos C sH sBrClz0 3PS = 366.0 (2104-96-3) Assay Contains not les s than 99.0% of C 4H\OO when
A suitable certified reference solution (10 ng/¡.¡.L in examined by the method for Benzene and other related
iso-octane) may be used. substances in the monograph for Isopropyl Alcohol.
Butan-2-oI sec-Butyl alcohol; 2-butanol;
Bromophos-ethyI ClOH IZBrClz03PS = 394.0
( 4824-78-6) C4 H\OO = 74.12 (78-92-2)
A suitable certified reference solution (lO ng/¡.¡.L in Analytical reagent grade of commerce.
iso-octane) may be used. A colourless liquid; boiling point, about 99°; d~g, about 0.81.
BromothymoI BIue 4,4 '-(3H-2,1-Benzoxathiol- Butan-2-oIRl See 2-Butanol R1
3-ylidene)bis(2-bromothymol) S,S-dioxide; Butan-2-one Methyl ethyl ketone; ethyl methyl ketone;
CZ7HzsBrzOsS = 624 (76-59-5) C 4 H sO = 72.11 (78-93-3)
Reddish-pink or brownish powder, practically insoluble in Chromatographic reagent grade of cornmerce.
water, soluble in ethanol (96%) and in dilute solutions of A colourless, fiarnmable liquid; boiling point, about 79°;
alkali hydroxides. d~g, about 0.81.
BromothymoI BIue SoIution Rl ButyI Acetate C 6H 1Z O Z = 116.2 (123-86-4)
Dissolve 50 mg of bromothYl11ol blue in a mixture of 4 mL of Clear, colourless liquid, fiammable, slightIy soluble in water,
0.02M sodium hydroxide and 20 mL of ethanol (96%) and miscible with ethanol (96%).
d~g, about 0.88; n~, about 1.395.
COl11plies with the following tests. Distillation range Not les s than 95% distils between 123°
Sensitivity A mixture of 0.3 mL of the solution and and 126°.
100 mL of carbon dioxide-free water is yellow. Not more than ButyI Acetate Rl
0.1 mL of 0.02M sodiul11 hydroxide VS is required to change
the colour of the solution to blue. Content, minimum 99.5%, determined by gas
chromatography.
Colour change pH 5.8 (yellow) to pH 7.4 (blue).
Clear, colourless liquid, fiammable, slightIy soluble in water,
BromothymoI BIue SoIution R2 A 1% w/v solution of miscible with ethanol (96%) .
bromothymol blue in dimethylformal11ide.
d~g' about 0.883; n~, about 1.395.
Bromothymo1 BIue Solution R3 Warm 0.1 g of
bromothymol blue with 3.2 mL of 0.05M sodium hydroxide and Butanol, maximum 0.2%, determined by gas
5 mL of ethanol (90%) . After solution is effected, add chromatography.
sufficient ethanol (90%) to produce 250 mL. n-Butyl formate, maximum 0.1%, determined by gas
BromothymoI BIue SoIution R4 Dissolve 100 mg of chromatography.
bromothymol blue in a mxture of equal volumes of ethanol n-Butyl propionate, maximum 0.1 %, determined by gas
(96%) and water and dilute to 100 mL with the same chromatography.
mixture of solvents . Filter if necessary. Water, maximum 0.1%.
BRP Indicator SoIution Dissolvl! 0.1 g of bromothymol Buty1amine n- Butylamine, 1-butanamine;
blue, 20 mg of l11ethyl red and 0.2 g of phenolphthalein in C 4H ll N = 73.1 (109-73-9)
sufficient ethanol (96%) to produce 100 mL. Filter. Distil and use within one month.
Brucine 10, 11-Dimethoxystrychnine; Colourless liquid, miscible with water, with ethanol (96%) .
CZ3Hz6Nz04,2HzO = 430.5 (357-57-3)
n~, about 1.401.
Colourless crystals, slightly soluble in water, freely soluble in Boiling point, about 78°.
ethanol (96%).
n-ButyIamine See Butylamine.
tert- Butylamine See 1, 1-Dimethylethylamine. Caffeic Acid 3,4-Dihydroxycinnamic acid,

Butylated Hydroxyanisole 2-tert- Butyl-4-methoxyphenol; (E)-3-(3,4-Dihydroxyphenyl)propenoic acid;
CllH¡602 = 180.2 (25 013-16-5) C 9 H S0 4 = 180.2 (331-39-5)
General reagent grade of commerce. White or almost white crystals or plates, freely soluble in hot
water and in ethanol (96%), sparingly soluble in cold water.
A white or almost white, crystalIine powder; melting point,
about 61 °. M elting point, about 225°, with decomposition.
Butylated Hydroxytoluene See Butylhydroxytoluene Absorbance (2.2.25) . A freshly prepared solution at
pH 7.6 shows 2 absorption maxima at 293 nm and 329 nm.
Butylboronic Acid C 4 H¡¡B0 2 = 101.9 (4426-47-5)
Caffeine CgHI ON40 2 = 194.2 (58-08-2)
Content, minimum 98%.
Of the British Pharmacopoeia.
Melting point, 90° to 92°.
Calciferol Ergocalciferol; vitamin D 2;
Butyl Chloride See 1-Chlorobutane.
C 2s H 44 0 = 396.7 (50-14-6)
tert-Butylhydroperoxide
Crystalline reagent grade of commerce.
1,I-Dimethylethylhydroperoxide; C 4 H IO 0 2 = 90.1 (75-91-2)
Colourless crystals or a white, crystalline powder; melting
Flammable liquid, soluble in organic solvents.
point, about 117°; [al~o, about + 105 (4% w/v in ethanol).
d~g, about 0.898; n~o, about 1.401; boiling point, 35°.
Calcium Acetate, Dried C 4 H 6 0 4 Ca = 158 .2 (62-54-4)
Butyl 4-hydroxybenzoate See Butyl parahydroxybenzoate.
Butylhydroxytoluene Butylated Hydroxytoluene of the
Calcium Carbonate CaC0 3 = 100.1 (471-34-1)
British Pharmacopoeia.
Butyl Parahydroxybenzoate Butyl Hydroxybenzoate of Of the British Pharmacopoeia .
the British Pharmacopoeia. Calcium Carbonate Rl
tert-Butyl Methyl Ether See 1, 1-Dimethylethyl methyl Complies with the requirements prescribed for calcium
ether. carbonate with the folIowing additional requirement.
Butyl Methacrylate Butyl 2-methylpropenoate; Chlorides (2.4.4) : maximum 50 ppm.
CSH¡402 = 142.2 (97-88-1) Calcium Chloride CaCI 2,2H 20 = 147.0 (10035-04-8)
Clear, colourless solution. Calcium Chloride Dihydrate of the British Pharmacopoeia.
d¡O, about 0.894; n~o, about 1.424; boiling point, about Calcium Chloride, Anhydrous
163°. CaCI2 = 111.0 (10043-52-4)
Butyric Acid n-Butyric acid; butanoic acid; Content, minimum 98.0% (dried substance).
C 4 H s0 2 = 88.1 (107-92-6) White or almost white granules, deliquescent, very soluble in
Content, minimum 99.0%. water, fre ely soluble in ethanol (96%) and in methanol.
Oily liquid, miscible with water and with ethanol (96%). Loss on drying (2.2.32): maximum 5.0%, determined by
d~g, about 0.96; n~o, about 1.398; boiling point, about 163". drying in an oven at 200°.
Butyrolactone Dihydro-2(3H)-furanone; y-butyrolactone; Store in an airtight container, protected from moisture .
C 4H 60 2 = 86.1 (96-48-0) Flammable liquid, soluble in Calcium Chloride Rl Calcium chloride tetrahydrate;
organic solvents. CaClz,4H 20 = 183.1
Oily liquid, miscible with water, soluble in methanol. General reagent grade of commerce containing not more
nfS' about 1.435; boiling point, about 204°. than 0.05 ppm of Fe.
Cadmium Cd = 112.4 (7440-43-9) Calcium Chloride Solution A 7.35% w/v solution of
ealcium ehloride.
Silvery-white, lustrous metal, practicalIy insoluble in water,
freely soluble in nitrie acid and hot hydroehlorie acid. Calcium Chloride Solution, O.OlM Dissolve 0.147 g of
caleium ehloride in water and dilute to 100.0 mL with the
Cadmium Acetate
C 4 H 60 4Cd,2H 20 = 266.5 (5743-04-4) same solvent.
Analytical reagent grade of commerce. Calcium Chloride Solution, O.02M Dissolve 2.94 g of
ealcium ehloride in 900 mL of water, adjust to pH 6.0 to 6.2
Cadmium and Ninhydrin Solution Dissolve 50 mg of
and dilute to 1000 mL with water.
eadmium aceta te in a mixture of 5 mL of water and l mL of
glacial aeetie acid and dilute with butan-2-one to 50 mL. Store at 2° to 8°.
Immediately before use add and dissolve sufficient ninhydrin Calcium chloride solution, O.025M Dissolve 0.368 g of
to produce a solution containing 0.2% w/v. calcium chloride in water and dilute to 100.0 mL with the
Cadmium Iodide CdI2 = 366.2 (7790-80-9) same solvent.
Analytical reagent grade of commerce. Calcium Hydroxide Calcium dihydroxide;
Cadmium Iodide Solution A 5.0% w /v solution of Ca(OH)z = 74.1 (I305-62-0)
eadmium iodide. White or almost white powder, almost completely soluble in
Cadmium Nitrate Tetrahydrate 600 parts of water.
Cd(N0 3)z,4H 20 = 308.5 (10022-68-1) Calcium Hydroxide Solution A freshly prepared
Hygroscopic orthorhombic crystals, very soluble in water, saturated solution which may be prepared in the folIowing
soluble in acetone and in ethanol (96%). manner. Shake 10 g of calciwl1 hydroxide repeatedly with
Melting point, about 59.5°. 1000 mL of water and allow to stand until c1ear.
Caesium Chloride CsCI = 168.4 (7647-1 7-8) Calcium Lactate (41372-22-9)
White or almost white powder, very soluble in water, freely Calcium Lactate Pentahydrate of the British Pharmacopoeia.
soluble in methanol, practicalIy insoluble in acetone .
2014 Appendix 1 A V -A3 7
Calcium Phosphate Monobasic Monohydrate CaJcimn The content of capric acid is not les s than 98%, caJculated
tetrahydrogen bisphosphate monohydrate; Phosphoric acid by the normalisation procedure.
calcium salt (2: 1) monohydrate; Capric Alcohol See Deean-1-ol.
CaH 40SP 2,H 2 0 = 252.1 (10031-30-8) Caproic Acid Hexanoic acid;
White or almost white, crystalline powder, soluble in water. C 6H 120 2 = 116.2 (142-62-1)
Calcium Sulfate Calcimn sulfate hemihydrate, calcium Oily liquid, sparingly soluble in water. d¡O, about 0.926;
sulphate hemihydrate, caJcium sulphate; plaster of Paris; n~, about 1.417; boiling point, about 205°.
CaS04,Y2 H 2 0 = 145 .1 (10034-76-1) Caproie aeid used in the assay of total fatty acids in Saw palmetto
White or almost white powder, soluble in about 1500 parts fruit (1848) eomplies with the following additional test.
ofwater, practically insoluble in ethanol (96%). When mixed Assay Examine by gas chromatography (2.2.28) as
with half its mass of water it rapidly solidifies to a hard and prescribed in the monograph on Saw palmetto fruit (1848).
porous mass.
The content of caproic acid is not less than 98%, caJculated
Calcium Sulfate Solution Calcimn sulphate solution by the normalisation procedure.
Shake 5 g of ealcium sulfate with 100 mL of water for 1 hour E-Caprolactam Hexane-6-lactam;
and filter. C 6H l1 NO = 113.2 (105-60-2)
Calconcarboxylic Acid Patton and Reeder's reagent; Hygroscopic fiakes, freely soluble in water, in anhydrous
2-hydroxy-1-(2-hydroxy-4-sulfo-I-naphthylazo )naphthalene- ethanol and in methano!.
3-carboxylic acid; C21H14N207S,3H20 = 492.5 (3737-95-9)
Brownish-black powder, slightly soluble in water, very slightly
Capryl Alcohol See Deean-1-01.
soluble in acetone and in ethanol (96%), sparingly soluble in
dilute solutions of sodium hydroxide. Caprylic Acid Octanoic acid;
C SH 16 0 2 = 144.2 (124-07-2)
Calconcarboxylic Acid Triturate Mix 1 pan of
ealeonearboxylie aeid with 99 pans of sodium ehloride. Slightly yellow, oily liquido d¡O, about 0.910; n~, about
1.428; boiling point, about 239.7°; melting point, about
Test/or sensitivity Dissolve 50 mg of caJconecarboxylic
16.7°.
acid triturate in a mixture of 2 mL of strong sodium hydroxide
solution and 100 mL of water. The solution is blue but Caprylie acid used in the assay of total fatty acids in Saw
becomes violet on addition of 1 mL of a 1.0% w/v solution palmetto fruit (1848) complies with the following additional test.
of magnesium sulfate and 0.1 mL of a 0.15 % w/v solution of Assay Examine by gas chromatography (2.2.28) as
ealeium ehloride and turns pure blue on addition of 0.15 mL prescribed in the monograph on Saw palmetto fruit (1848) .
of O. 01 M sodium edetate. The content of caprylic acid is not less than 98%, caJculated
Camphene 2,2-Dimethyl-3-methylenebicyclo [2 .2.1] by the normalisation procedure.
heptane; C 1oH 16 = 136.2 (79-92-5) Capsaicin (E) -N-[ (4-Hydroxy-3-methoxyphenyl)methyl]-
Camphene used in gas ehromatography complies with the following 8-methylnon-6-enamide; C18H27N03 = 305.4 (404-86-4)
additional test. White or almost white, crystalline powder, practically
Assay Carry out the method for Chromatographic profile insoluble in water, freely soluble in anhydrous ethano!.
described in the monograph for Rosemary Oil. The content Melting point, about 65°.
is not les s than 90% caJculated by the normalisation
Capsaicin used in the assay in Capsieum (1859) complies with
procedure.
the following additional test.
Camphor Racemic camphor; C lO H 16 0 = 152.2 (76-22-2) Assay Liquid chromatography (2.2.29) as prescribed in the
Racemic Camphor of the British Pharmacopoeia. monograph Capsieum (1859) .
(lS)-(+ )-lO-Camphorsulfonic Acid (1S)-(+)-10- Content, minimum 95.0%, caJculated by the normalisation
Camphorsulphonic Acid; (lS,4R)-( + )-2-0xo-1 0- procedure.
bornenesulfonic acid, [( 1S)-7,7 -Dimethyl-2- Carbazole Dibenzopyrrole; C 12H 9 N = 167 .2 (86-74-8)
oxobicyclo [2.2. 1] heptan-1-yl]methanesulfonic acid;
Crystals, practically insoluble in water, freely soluble in
Reychler's acid; C lO H 16 0 4S = 232.3 (3144-16-9)
acetone, slightly soluble in anhydrous ethano!.
Prismatic crystals, hygroscopic, soluble in water.
Content, minimum 99.0% of (1S)-(+ )-10-camphorsulfonic
acid. Carbomer (9007-20-9) A cross-linked polymer of acrylic
acid; it contains a large proponion (56% to 68%) of
[a]~o, + 19 to +21 (4.3% w/v solution in water). carboxylic acid (COOH) groups after drying at 80° for
Melting point, about 194°, with decomposition. 1 hour. Average relative molecular mass about 3 x 10 6.
M (2.2.41): 10.2 x 10 3 determined at 290.5 nm on a pH (2.2.3): about 3 for a 1.0% w/v suspension.
0.10% w/v solution. Carbon Dioxide CO 2 = 44.01 (124-38-9)
Capric Acid Decanoic acid; Of the British Pharmacopoeia.
C lOH 20 0 2 = 172.3 (334-48-5)
Carbon Dioxide Rl
Crystalline solid, very slightly soluble in water, soluble in
ethano!. Boiling point, about 270°; melting point, about Content, minimum 99.995% v/v.
31.4°. Carbon monoxide, les s than 5 ppm.
Caprie acid used in the assay of total fatty acids in Saw palmetto Oxygen, less than 25 ppm.
fruit (1848) eomplies with the following additional test. Nitric oxide, less than 1 ppm.
Assay Examine by gas chromatography (2.2.28) as Carbon Dioxide R2 Carbon dioxide containing not less
prescribed in the monograph on Saw palmetto fruit (1848). than 99% v/v of CO 2.
Carbon Disulfide Carbon disulphide; Carvacrol used in gas chromatography complies with the following
CS 2 = 76.14 (75-15-0) additional test.
Colourless or yeIlowish, fiammable liquid, practicalIy Assay Carry out the method described in the monograph
insoluble in water, miscible with anhydrous ethano!. for Pepperrnint Oi!.
dig, about 1.26; boiling point, 46° to 47°. Test solution Dissolve 0.1 g in about 10 mL of acetone.
Carbon for Chromatography, Graphitised Carbon Content, minimum 95 .0%, caIculated by the normalisation
chains having a length greater than C 9 with a particle size of procedure.
400 to 850 ~lm. Carveo1 p-Mentha-1 (6),8-dien-2-01; 2-Methyl-
D ensity, 0.72; surface are a, 10 m Z g- l. 5-( 1-methylethenyl) cyclohex-2-enol;
Do not use at a temperature higher than 400°. C lO H 160 = 152.2 (99-48-9)
Carbon for Chromatography, Graphitised Rl Porous The substance contains a variable content of trans- and
spherical carbon particles comprised of fiat sheets of cis-carveo!.
hexagonaIly arranged carbon atoms. Carveol used in gas chromatographycomplies with the following
Particle size 5-7 ¡.tm; Pore volume 0.7 cm 3/g. additional test.
Carbon Monoxide CO = 28.01 (630-08-0) Assay Gas chromatography (2.2.28) as prescribed in the
test for chromatographic profile in the monograph on
Caraway oi!.
99 .97% v/v of CO.
Content, minimum 97%, caIculated by the normalisation
Carbon Monoxide Rl CO = 28.0 1 (630-08-0)
procedure.
Contains not less than 99 % v/v of CO.
Carvone (+ )-p-Mentha-6,8-dien-2-one; (5S)-2-Methyl-
Carbon Tetrach10ride Tetrachloromethane; 5-( 1-methylethenyl)-cyclohex-2-enone;
CCI 4 = 153.8 (56-23-5) C lO H 14 0 = 150.2 (2244-16-8)
Clear, colourIess liquid, practicaIly insoluble in water, Liquid, practicaIly insoluble in water, miscible with ethanol
miscible with ethanol (96%). (96%).
dig, 1.595 to 1.598; boiling point, 76° to 77° dig, about 0.965; n~o, about 1.500; [al~o, about + 61; boiling
Carbophenothion 0,0-Diethyl point, about 230°.
s-{[(4-chlorophenyl)thio] methyl}phosphorodithioate; Carvone used in gas chromatography complies with the following
CllH16CIOzPS3 = 342.9 (786-19-6) additional test.
YeIlowish liquid, practicaIly insoluble in water, miscible with Assay Gas chromatography (2.2.28) as prescribed in the
organic solvents. monograph Peppermint oil (0405) using the substance to be
d¡5, about 1.27 . examined as the test solution.
For the monograph Wool Fat (0134), a suitable certified Content, minimum 98.0%, caIculated by the n ormalisation
reference solution (10 ng/¡.tl in iso-octane) may be used. procedure.
Car-3-ene 3,7, 7-Trimethylbicyclo[ 4.1.0]hept-3-ene; Carvone Rl (2244-16-8)
4,7,7-trimethyl-3-norcarene; ClOH1 6 = 136.2 (498-15- 7) Complies with the requirements prescribed for carvone with the
Liquid with a pungent odour, slightIy soluble in water, following additional requirement.
soluble in organic solvents . Assay Gas chromatography (2.2.28) as prescribed in the
d~g, about 0.864; n~o, 1.473 to 1.474; [al~o, + 15 to + 17; test for chiral purity in the monograph on Caraway oil
boiling point, 170° to 172°. (1817) .
Car-3-ene used in gas chromatography complies with the following Content, minimum 98%.
additional test.
(- )-Carvone (- )-p-Mentha-1(6),8-dien-2-one;
Assay Gas chromatography (2.2.28) as prescribed in the (5 R)- 2-Methyl-5-( l-methylethenyl) cyclohex-2-enone;
monograph Nutmeg oil (1552). C lOH 14 0 = 150.2 (6485-40-1 )
Content, minimum 95.0%, caIculated by the normalisation Liquid.
procedure.
dig, about 0.965; n~o , about 1.4998; [al~o, about -62; boiling
Carminic Acid 7 -ex-D-Glucopyranosyl-3,5,6,8- point, about 230°.
tetrahydroxy-1-methyl-9,1 0-dioxo-9, 10-dihydroanthracene-
Assay Gas chromatography (2.2.28) as prescribed in the
2-carboxylic acid; CzzHzo013 = 492.4 (1260-1 7-9) test for chiral purity in the monograph on Caraway oi!.
Dark red powder, very slightIy soluble in water, soluble in
Content, mínimum 99%.
dimethyl sulfoxide, very slightly soluble in ethanol (96%).
B-Caryophyllene (E)-(lR,9S)-4, 11, 11-Trimethyl-
Carob Bean Gum Locust bean gum 8-methylenebicyclo [7 .2.0]undec-4-ene;
The ground endosperm of the fruit kernels of Ceratonia C 1sH z4 = 204.4 (87-44-5)
süiqua L. T aub .
Oily liquid, practicaIly insoluble in water, miscible with
White or almost white powder containing 70% to 80% of a ethanol (96%).
water-soluble gum consisting mainly of galactoma=oglycone.
B-Caryophyllene used in gas chl'omatography complies withthe
Carvacro1 5-Isopropyl-2-methylphenol; following additional test.
C lOH 140 = 150.2 (499-75-2)
Assay. Gas chromatography (2.2.28) as prescribed in the
Brownish liquid, practicalIy insoluble in water, very soluble in monograph Clove oil (1091).
ethanol (96%).
Test solution The substance to be examined.
d~g, about 0. 975; n~, about 1.523; boiling point, about
Content, minimum 90.0%, caIculated by the normalisation
23T.
procedure.
CaryophyIlene Oxide (-)-~-Caryophyllene epoxide; polymer lattice consisting of polystyrene cross-linked with
(lR,4R,6R, I 0S)-4,12, 12-Trimethyl-9-methylene-5- 8% of divinylbenzene. The particle size is specified after the
oxatricyclo [8 .2. O. 04,6Jdodecane; name of the reagent in tests where it is used.
C lsH 240 = 220.4 (1139-30-6) Cation Exchange Resin (Sodium Form), Strong Resin
Colourless, fine crystals with lumps. Melting point, 62' to in sodium form with sulfonic acid groups attached to a
63°. polymer lattice consisting of polystyrene cross-linked with
Ca/yophyllene oxide used in gas chromatography complies with the divinylbenzene. The parricle size is specified after the name
following additional test. of the reagent in the tests where it is used.
Assay Examine by gas chromatography (2.2.28) as Cation Exchange Resin Rl A resin in protonated form
prescribed in the monograph on Turpentine oil, Pinus pinaster with sulfonic acid groups attached to a polymer lattice
type. consisting of polystyrene cross-linked with 4% of
divinylbenzene. It is available as beads and the particle size is
The content is not less than 99.0%, calculated by the
specified after the name of the reagent in tests where itis
nonnalisation procedure .
used.
Casein (9000-71-9) Mixture of related phosphoproteins
Cationic Resin, Weak Polymethacrylic resin
obtained from milk.
A weak cationic res in, slightly acidic with carboxyl groups
White or almost white, amorphous powder or granules, very
present in a protonated form, of particle size 75 to 160 )..lm.
slightly soluble in water and in non-polar organic solvents. It
dissolves in concentrated hydrochloric acid giving a pale- The res in should be used within the pH limits of 5 to 14 and
should not be used at temperatures aboye 120 0 •
violet solution. It forms salts with acids and bases.
Its isoelectric point is at about pH 4.7 . Alkaline solutions are Cedarwood Oi! A grade of commerce for microscopy
laevorotatory. thickened as necessary in temperate or tropical climates.
Casein Substrate, Concentrated Suspend a quantity of Cellulose See Cellulose for chromatography.
casein EPBRP equivalent to 2.5 g in 5 mL of water, add Cellulose for Chromatography (9004-34-6) Fine, white
18 mL of O.IM sodiwn hydroxide and stir for I minute. Add or almost white, homogeneous powder with an average
60 mL of water and stir with a magnetic stirrer until the particle size less than 30 )..lm.
solution is practically clear. Adjust the pH of the solution to Preparation 01 a thin layer. Suspend 15 g in 100 mL of
8.0 with either O.l lvl sodium hydroxide or O.IM hydrochloric acid water and homogenise in an electric mixer for 60 seconds.
and add sufficient water to produce 100 mL. Coat carefully cleaned plates with a layer 0.1 mm thick using
Use on the day of preparation. a spreading device. Allow to dry in air.
Casticin 5-Hydroxy-2-(3-hydroxy-4-methoxyphenyl)- Cellulose for Chromatography Rl Microcrystalline
3,6,7-trimethoxy-4H-l-benzopyran-4-one; cellulose
Cl 9Hl SOS = 374.3 (479-91-4) A fine, white or almost white homogeneous powder with an
Yellow crystals. average particle size less than 30 )..!ffi.
Catalpol (1aS, lbS, 2S,5 aR, 6S, 6aS)-6-H ydroxy- Preparation 01 a thin-layer Suspend 25 g in 90 mL of
I a-hydroxymethyl)-1 a, 1b,2,5a,6,6a- water and homogenise in an electric mixer for 60 seconds.
hexah ydrooxireno [4 ,5J cyclopenta [1 ,2-cJpyran-2-yl Coat carefully cleaned plates with a layer O.I-mm thick using
~-D-glucopyranoside; Cl5H2201O = 362.3 (2415-24-9) a spreading device. Allow to dry in airo
0
Melting point, 203 to 205 ' . Cellulose for Chromatography F 254 Microcrystalline
Catechin (+ )-(2R,3S)-2-(3,4-Dihydroxyphenyl)-3,4- cellulose F 254 . A fine, white or almost white, homogeneous
dihydro-2H-chromene-3,5, 7-triol, Catechol, Cianidanol, powder with an average parricle size less than 30 ~lm,
Cyanidol; C15H1406,xH20 = 290.3 (anhydrous) (154-23-4) containing a fiuorescent indicator having an optimal intensity
at 254 nm.
Catechol Pyrocatechol; benzene-l,2-diol; Preparation 01 a thin layer Suspend 25 g in 100 mL of
water and homogenise using an electric mixer for 60 seconds.
C 6H 60 2 = 110.1 (120-80-9)
Coat carefully cleaned pI ates with a layer 0.1 mm thick using
General reagent grade of commerce. a spreading device. Allow to dry in airo
0
A white, crystalline powder; melting point, about 103 •
Cellulose F 254 See Cellulose for chromatography F254 •
Store protected from light. Cellulose, MicrocrystaIline See Cellulose for
Catholyte for Isoelectric Focusing pH 3 to 5 chro711atography R 1.
(O.IM ~-Alanine) Dissolve 8.9 g of alanine in sufficient water Cephaeline Dihydrochloride (R)-I-[(2S,3R, 11 bS)-
to produce 1000 mL. 3-Ethyl-l,3,4,6,7 ,11 b-hexahydro-9, 10-dimethoxy-2H-
Cation Exchange Resin A resin in protonated form with benzo [a JquinolizinylmethylJ -1 ,2,3,4-tetrahydro-7-
sulfonic groups attached to a polymer lattice consisting of methoxyisoquinolin-6-01 dihydrochloride heptahydrate;
polystyrene cross-linked with 8% of divinylbenzene. It is C2sH40C12N 20 4,7H 20 = 666 (5884-43-5)
available as beads and the parricle size is specified after the General reagent grade of commerce.
name of the reagent in tests where it is used.
A white to yellowish, crystalline powder; [ClJ~O , about +25
Cation-Exchange Resin, Strong Strong cation-exchange (2% w/v in water).
resin in protonated form with sulfonic acid groups attached
Cephalin Reagent Solvents used to prepare this reagent
to a polymer lattice consisting of polystyrene cross-linked
with divinylbenzene. The particle size is specified after the should contain a suitable antioxidant such as butylated
hydroxyanisole at a concentration of 0.002 % w/v.
name of the reagent in the tests where it is used.
Cation Exchange Resin (Calcium Form), Strong Resin To 0.5 to 1 g of acetone-dlied ox brain add 20 mL of acetone
and leave for 2 hours. Centrifuge for 2 minutes at 500 g and
in calcium fonn with sulfonic acid groups attached to a
decant the supernatant liquido Dry the residue under reduced Chloral Hydrate Solution Dissolve 80 g of ehloral hydrate
pressure and extract the dried material with 20 mL of in 20 mL of water.
ehloroform for 2 hours, shaking the mixture trequendy. After Chloramine See Chloramine T.
removal of the solid material by filtration or centrifugation, Chloramine Solution A 2% w/v solution of ehloramine T
evaporate the chloroform trom the extract under reduced prepared immediately before use.
pressure. Suspend the residue in 5 to 10 mL of saline solution.
This stock emulsion may be stored frozen or freeze-dried for Chloramine Solution Rl A 0.01 % w/v solution of
3 months. ehloramine T prepared immediately before use.
Cerium(m) Nitrate Cerous nitrate, Cerium trinitrate Chloramine Solution R2 A 0.02% w/v solution of
hexahydrate; Ce(N03h,6H 20 = 434.2 (10294-41-4) ehloramine T prepared immediately before use.
Colourless or pale yellow, crystalline powder, freely soluble in Chloramine T Chloramine; the sodium salt of
water and in ethanol (96%). N-chlorotoluene-p-sulfonamide;
C7H7CINNa02S,3H20 = 281.7 (55-86-7)
Cerium(m) Nitrate Solution Dissolve 0.22 g of
eerium(IlI) nitra te in 50 mL of water, add 0.1 mL of nitrie acid Tosylchloramine Sodium of the British Pharmacopoeia.
and 50 mg of hydroxylamine hydroehloride and dilute to Chlordane ClOH6Cla = 409.8 (12789-03-6)
1000 mL with water. Boiling point, about 175°; melting point, about 106°.
Cerium Sulfate Ceric sulfate, ceric sulphate, cerium(Iv) A suitable certified reference solution of technical grade
sulfate tetrahydrate; Ce(S04h,4H 20 = 404.3 (10294-42-5) (lO ng/)lL in iso-octane) may be used.
Appearance, yellow or orange-yellow, crystalline powder or Chlordiazepoxide C I6H 14 CIN30 = 299.8 (58-25-3)
crystals. Of the British Pharmacopoeia.
Solubiliry, very slighdy soluble in water, slowly soluble in Chlorfenvinphos C12H14C1304P = 359.6 (470-90-6)
dilute acids.
A suitable certified reference solution (10 ng/)lL in
Cerium(IV) Sulfate See Cerium sulfate. cyclohexane) may be used.
Cerous Nitrate See Cenum(IlI) nitrate. Chlorhexidine Acetate Chlorhexidine diacetate;
Cetostearyl Alcohol Of the British Pharmacopoeia. C22H 30C12NIO,2C2H402 = 625.6 (56-95-1)
Cetrimide (8044-71-1) Of the British Pharmacopoeia. General reagent grade of commerce.
Cetyl Alcohol Hexadecan-l-ol; Melting point, about 154°.
C l6 H 340 = 242.4 (36653-82-4) Chlorhexidine Hydrochloride Chlorhexidine
Content, minimum 95.0% of C 16H 340. dihydrochloride; C22H30Cl2N 1O,2HCI (3697-42-5)
Melting point, about 48°. General reagent grade of commerce.
Cetylpyridinium Chloride Monohydrate Chloroacetanilide See 4'-Chloroaeetanilide.
1-Hexadecylpyridinium chloride monohydrate; 4' -Chloroacetanilide Chloroacetanilide;
C 21 H 3a CIN,H20 = 358.0 (6004-24-6) CaHaCINO = 169.6 (539-03-7)
White or almost white powder, freely soluble in water and in Content, minimum 95%.
ethanol (96%).
Crystalline powder, practically insoluble in water, soluble in
Melting point, 80° to 83°. ethanol (96%).
Cetyltrimethylarnmonium Bromide Cetrimonium Melting point, about 178°.
bromide; N-hexadecyl-N,N,N-trimethylammonium bromide;
Chloroacetic Acid C 2H 3CI0 2 = 94.50 (79-11-8)
C l9 H 42 BrN = 364.5 (57-09-0)
Colourless or white or almost white crystals, deliquescent,
White or almost white, crystalline powder, soluble in water,
very soluble in water, soluble in ethanol (96%) .
freely soluble in ethanol (96%).
Chamazulene 7-Ethyl-l,4-dimethylazulene; Chloroaniline See 4-Chloroaniline.
C14HI6 = 184.3 (529-05-5) 3-Chloroaniline C 6H 6CIN = 127.6 (108-42-9)
Blue liquid, very slightly soluble in water, soluble in ethanol General reagent grade of commerce.
(96%), miscible with fatty oils, with essential oils and with n~, about 1.594.
liquid paraffin, soluble with discolouration in phosphoric acid 4-Chloroaniline Chloroaniline;
(85% mlm) and sulfuric acid (50% v/v). C 6 H 6 CIN = 127.6 (106-47-8)
Appearance 01 solution 50mg is soluble in 2.5 mL of
Crystals, soluble in hot water, freely soluble in ethanol
hexane. The blue solution is clear in a thin-Iayer obtained by
(96%).
tilting the test-tube.
Melting point, about 71 0 .
Chamazulene used for gas ehromatography eomplies with the
following additional test. Chloroauric Acid Gold chloride; hydrogen
Assay Examine by gas chromatography as prescribed in tetrachloroaurate; HAuCI 4 + aq (27988-77-8)
the monograph on Matriciearia Dil, using a 4 gIL solution in General reagent grade of commerce.
eyclohexane. , Brown, deliquescent masses.
The content of chamazulene is not less than 95.0%, Chloroauric Acid Solution General reagent grade of
calculated by the normalisation procedure. commerce containing about 2% w/v of HAuCI 4 ,3H 20, or a
Charcoal, Activated (64365-11-3) 2.0% w/v solution of ehloroaurie aeid in water.
Of the British Pharmacopoeia. Chlorobenzene C 6 H sCI = 112.6 (108-90-7)
Chloral Hydrate C 2H 3CI 30 2 = 165.4 (302-17-0) Analytical reagent grade of cornmerce.
Of the British Pharmacopoeia. A colourless liquid; boiling point, about 131
0 .
4-Chlorobenzenesulfonamide Ethanol 0.4% w/w to 1.0% w/w.

4-Chlorobenzenesulphonamide; Introduce 1.00 g (m g) into a ground-glass-stoppered fiask.
C 6H 6CIN0 2 S = 191.6 (98-64-6) Add 15 .0 mL of nitrochromic reagent, c10se the fiask, shake
White or almost white powder. vigorously for 2 minutes and allow ro stand for 15 minutes .
Melting point, about 145°. Add 100 mL of water and 5 mL of a 200 gIL solution of
2-Chlorobenzoic Acid C 7 H sCI0 2 = 156.7 (118-91-2) potassium iodide. After 2 minutes titrate with 0.1 M sodium
thiosulfate, using 1 mL of starch solution as indicaror, until a
Soluble in water, slightly soluble in anhydrous ethano!. Iight green colour is obtained (n) mL of 0.1 M sodium
Boiling point, about 285°; melting point, about 140°. thiosulface). Carry out a blank assay (n2 mL of 0.1 M sodium
4-Chlorobenzoic Acid C 7H sCI0 2 = 156.57 (74-11-3) thiosulfate). Calculate the percentage of ethanol using the
General reagent grade of commerce. following expression :
Melting point, about 240 0 •
3-(4-Chlorobenzoyl)propionic Acid (n 2 -n,) 0.115
C lO H 9CIN0 3 = 212.6 (3984-34-7) 111

Melting point, about 129°. Chloroform, Acidified To 100 mL of chlorofor111 add
l-Chlorobutane Butyl chloride; 10 mL of hydrochloric acid. Shake, allow ro stand and
C 4 H 9C1 = 92.57 (109-69-3) separate the rwo layers.
General reagent grade of commerce. Chloroform, Ethanol-free Shake 200 mL of chloroform
with four quantities, each of 100 mL, of water. Dry over
Boiling point, about 78°; weight per mL, about 0.886 g;
20 g of anhydrous sodiw11 sulfate for 24 hours. Distil the
nbo, about 1.402. filtrate over 10 g of anhydrous sodium sulfate. Discard the first
Chlorobutanol Anhydrous chlorbutol; 1,1, l-trichloro- 20 mL of distillate. Prepare immediately before use.
2-methylpropan-2-01; C 4 H 7 CI 3 0 = 177.5 (57-15-8)
Chloroform IR
Anhydrous Chlorobutanol of the British Pharmacopoeia.
Spectroscopic reagent grade of commerce.
4-Chloro-o-cresol 4-Chloro-2-methylphenol;
Chloroform Stabilised with Amylene CHCI 3 = 119.4
C 7H 7 CIO = 142.6 (1570-64-5)
Clear, colourless liquid, slightly soluble in water, miscible
with ethanol (96%).
Water: maximum 0.05%.
2-Chloro-2-deoXY-D-glucose
Residue on evaporation: maximum 0.001 %.
C 6H))CIO s = 198.6 (14685-79-1)
Minimum transmittance (2.2.25) using water as
White or almost white crystalline, very hygroscopic powder,
compensation liquid : 50% at 255 nm, 80% at260 nm, 98%
soluble in water and in dimethyl sulfoxide, practically
at 300 nm.
insoluble in ethanol (96%).
Content, minimum 99.8% of CHCI3, determined by gas
l-Chloro-2,4-dinitrobenzene
chromatography.
C 6H 3 CIN 2 0 4 = 202.6 (97-00-7)
Chloroform Water Shake 2.5 mL of chloroform with
900 mL of water until dissolved and dilute ro 1000 mL with
Pale yellow crystals or crystalline powder; melting point, water.
about 51 °.
Chlorogenic Acid (1S,3R,4R,5R)-3-[(3,4-
2-Chloroethanol Ethylene chlorohydrin; Dihydroxycinnamoyl)oxy] -I,4,5-
C2 H s CIO = 80.5 (107-07-3) trihydroxycyclohexanecarboxylic acid;
Colourless liquid, soluble in ethanol (96%). C 16H) s09 = 354.3 (327-97-9)
dig, about 1.197; n~, about 1.442; boiling point, about White or almost white, crystalline powder or needles, freely
130°; melting point, about -89°. soluble in boiling water, in acetone and in ethanol (96%) .
2-Chloroethanol Solution Dissolve 125 mg of [a]b6, about - 35.2.
2-chloroethanol in propan-2-01 and dilute to 50 mL with the Melting point, about 208°.
same solvent. Dilute 5 mL of the solution to 50 mL with
Chromatography Examined under the conditions
propan-2-01.
described on Identification A in the monograph on
Chloroethylamine Hydrochloride 2-Chloroethanamine Belladonna leaf dry extract, standardised (1294), the
monohydrochloride; C 2 H 7 CI 2N = 116.0 (870-24-6) chromatogram shows only one principal zone.
Melting point, about 145°. Chlorogenic acid used in liquid chromatography complies with the
(2-Chloroethyl)diethylamine Hydrochloride following additional test.
2-Diethylaminoethyl chloride hydrochloride; . Assay Liquid chromatography (2.2.29) as prescribed in the
C6H)4CIN,HCI = 172.1 (869-24-9) monograph on Artichoke Leaf (1866).
White or almost white, crystalline powder, very soluble in Content, minimum 97.0%.
water and in methanol, freely soluble in methylene chloride,
practically insoluble in hexane. 5-Chloro-8-hydroxyquinoline 5-Chloroquinolin-8-ol;
C 9H 6CINO = 179 .6 (130-16-5)
Mel ting point, abou t 211 °.
Chloroform Trichloromethane; CHCI 3 = 119.4 (67-66-3) General reagent grade of commerce.
Clear, colourless Iiquid, slightly soluble in water, miscible Melting point, about 123°.
with ethanol (96%). 3-Chloro-2-methylaniline 6-Chloro-2-toluidine;
dig, 1.475 to 1.481; boiling point, about 60°. C 7 H sCIN = 141.6 (87-60-5)
Not miscible with water, slightly soluble in anhydrous 4-Chlorosulfamoylbenzoic Acid

ethano!. C 7H 6CIN0 4 S = 235.7 (1205-30-7)
d;g, about 1.171; n5°, 1.587; boilingpoint, about 115°; General reagent grade of commerce.
melting point, about 2°. Chlorothiazide C7H6CIN304S2 = 295.7 (58-94-6)
2-Chloro-N- (2 ,6-dimethylphenyl)acetamide Of the British Pharmacopoeia.
C lOH 12CINO = 197.7 (1131-01-7) ChlorotrimethyIsilane See Tn·methylehlorosilane.
2-Chloronicotinic Acid 2-Chloropyridine-3-carboxylic Chlorotripheny1methane Tripheny1chloromethane;
acid; C 6H 4 CIN0 2 = 157.6 (2942-59-8) triphenylmethyl chloride; C 19H 1S CI = 278.8 (76-83-5)
White or almost white powder. General reagent grade of commerce.
Melting point, about 177°; content, minimum 95 %. A pale yellow or buff, crystalline sol id; melting point, about
2-Chloro-4-nitroaniline 112°.
C 6H sCIN 20 2 = 172.6 (121-87-9) Chlorpyriphos C 9H l l ChN0 3PS = 350.6 (2921-88-2)
Yellow, crystalline powder, freely soluble in methano!. Boiling point, about 200°; melting point, 42° to 44°.
Melting point, about 107°. A suitable certified reference solution (10 ng/¡.tL in
Store protected from light. cyc1ohexane) may be used.
2-Chloro-5-Nitrobenzoioc Acid Chlorpyriphos-methy1
C 7H 4 N0 4 CI = 201.6 (2516-96-3) C 7H 7CI3N0 3PS = 322.5 (5598-13-0)
Melting point, 165° to 168°. Melting point, 45° to 47°.
Chlorophenol See 4-Chlorophenol. A suitable certified reference solution (10 ng/¡.tL in
4-Chlorophenol Chlorophenol; cyc1ohexane) may be used.
C 6H sCIO = 128 .6 (106-48-9) Chlortetracycline Hydroch1oride Of the British
Colourless or almost colourless crystals, slightly soluble in Pharmacopoeia.
water, very soluble in ethanol (96%) and in solutions of alkali (5a)-Cho1estane C 27 H 48 = 372.7 (481-21-0)
hydroxides. Slightly soluble in anhydrous ethanol; melting point, about
Melting point, about 42°. 81 °.
Chloroplatinic Acid See Chloroplatinie(IV) aeid. Cho1estero1 Of the British Pharmacopoeia.
Chloroplatinic(rv) Acid Chloroplatinic acid; platinic Cho1estery1 Benzoate 5-Cholesten- 3~ -ol-3-benzoate;
chloride; H 2PtCI6 + aq (18497-13-7) C34H so0 2 = 490.8 (604-32-0)
Content, minimum 37.0% mlm ofplatinum (Ar 195.1 ). General reagent grade of commerce.
Brownish-red crystals or a crystalline mass, very soluble in Melting point, about 150°.
water, soluble in ethanol (96%). Cho1ine Chloride (2-Hydroxyethyl)trimethylammonium
Assay Ignite 0.200 g to constant mass at 900 ± 50° and chloride; C SH 14 CINO = 139.6 (67-48-1)
weigh the residue (platinum). Deliquescent crystals, very soluble in water and in ethanol
Store protected from light. (96%).
Chloroplatinic Acid Solution A solution of Chromatography Thin-Iayer chromatography (2.2.27) as
ehloroplatinic(IV) aeid in water, containing the equivalent of prescribed in the monograph Suxamethonium ehloride (0248):
5.0% w/v of H 2PtCl6,6H 20 . apply 5 ¡.tL of a 0.02% w/v solution in methanol;
3-Chloropropane-l,2-dioI C 3H 7CI0 2 = 110.5 (96-24-2) the chromatogram shows one principal spot.
Colourless liquid, soluble in water and ethanol (96%). Store in an airtighi: container.
d;g, about 1.322; n5°, about 1.480; boiling point, about Chondroitinase ABC Pectin Iyase-like enzyme secreted
213°. by Flavobacterium heparinum. Available in vials containing
5-Chloroquinolin-8-o1 5-Chlorooxine; 5-10 units. It c1eaves both glucuronate-containing
C 9H 6CINO = 179 .6 (130-16-5) disaccharides, e.g. chondroitin sulfate, and iduronate-
Sparingly soluble in cold dilute hydrochloric acid. containing disaccharides, e.g. dermatan sulfate.
Melting point, about 123°. Chondroitinase AC Pectin Iyase-like enzyme secreted by
Flavobacterium heparinum. Available in vials containing 5-10
Content, mínimum 95.0% . units. It c1eaves only glucuronate-containing disaccharides,
l-ChloropropyI (dimethylamine) Hydrochloride e.g. chondroitin sulfate.
3-Dimethylaminopropyl chloride hydrochloride; Chromazuro1 S See Chrome Azurol S.
C SH 12CIN = 158.1 (5407-04-5)
Chrome Azuro1 S CI 43825; trisodium 5-[(3-carboxylato-
General reagent grade of commerce. 5-methyl-4-oxocyc1ohexa-2,5-dien-1-ylidene) (2,6-dichloro-
Melting point, about 142°. 3-sulfonatophenyl)methyl]-2-hydroxy-3-methylbenzoate;
4-ChlororesorcinoI 4-Chlorobenzene-1,3-diol, C23H1 3ClzNa30 9S = 605 (1667-99-8)
1,3-dihydroxy-4-chlorobemene; . Brownish-black powder, soluble in water, slightly soluble in
C 6H sCI0 2 = 144.6 (95-88-5) ethanol (96%).
Melting point, 106° to 108°. Chromic Acid Cleansing Mixture See Chromic-sulfuric
5-Chlorosalicylic Acid C 7H sCI0 3 = 172.6 (321-14-2) acid mixture.
White or almost white, crystalline powder, soluble in Chromic Potassium Sulfate See Chromium(m) potassium
methano!. sulfate.
Chromic-Sulfuric Acid Mixture Chromic-sulphuric acid A yellowish-white powder, soluble in water, practically
mixture insoluble in ethanol (96%).
A saturated solution of ehromium(v¡) oxide in sulfurie aeid. Chromotropic Acid, Sodium Salt Solution Dissolve
Chromium(m) Acetylacetonate 0.60 g of ehromotropie acid, sodium salt in about 80 mL of
ClsH21Cr06 = 349.9 (21679-31-2) water and dilute to 100 mL with the same solvent. Use the
solution within 24 hours.
Chromium(VI) Oxide Chromium trioxide;
Cr03 = 100.0 (1333-82-0) Chromotropic Acid Solution Dissolve 0.5 g of
ehromotropie acid in sufficient water to produce 100 mL.
Dark brownish-red needles or granules, deliquescent, very
soluble in water. Use the solution within 24 hours .
Store in an airtight glass container. Chromotropic Acid-Sulfuric Acid Solution
Chromotropic Acid-Sulphuric Acid Solution
Chromium(m) Potassium Sulfate Chrome alum,
chromic potassium sulfate, chromic potassium sulphate, Dissolve 5 mg of ehromotropie acids sodium salt in 10 mL of a
chromium(m) potassium sulphate; mixture of 9 mL of sulfurie acid and 4 mL of water.
CrK(S04bI2H20 = 499.4 (7788-99-0) Chrysanthemin Cyanidin 3-0-glucoside chloride;
Large, violet-red or black crystals, freely soluble in water, Kuromanin Chloride; 2-(3,4-Dihydroxyphenyl)-3-(P-D-
practically insoluble in ethanol (96%). glucopyranosyl)oxy-5, 7-dihydroxy-l-benzopyrylium chloride;
C21H21CIOll = 485.8 (7084-24-4)
Chromium(m) Trichloride Hexahydrate
[Cr(H20)4Clz]CI,2H20 = 266.5 (10060-12-5) Reddish-brown crystalline powder, soluble in water and in
ethanol (96%).
Dark green crystalline powder, hygroscopic.
Absorbance A 0.001 % w/v solution in a mixture of
Store protected from humidity and oxidising agents.
1 volume of hydroehlorie acid and 999 volumes of methanol
Chromium Trioxide See Chromium(v¡) oxide. shows a maximum at 528 nm.
Chromogenic Substrate Rl Dissolve N-a- a.-Chymotrypsin for Peptide Mapping a.-Chymotrypsin
benzyloxyearbonyl-D-arginyl-L-glyeyl-L-arginine-4- of high purity, treated to eliminate tryptic activity.
nitroanilidedihydroehloride in water to give a 0.003M solution.
Cimifugin (2S) -7 -(Hydroxymethyl)-2-( l-hydroxy-l-
Dilute in tris(hydroxymethyl)aminomethane-EDTA buffer
methylethyl)-4-methoxy-2,3-dihydro-5H-furo[3,2-
solution pH 8.4to 0.0005 M before use.
g][l]benzopyran-5-one; C16HlS06 = 306.3 (37921-38-3)
Chromogenic Substrate R2 Dissolve D-phenylalanyl-L-
Cinchonidine (R)-(Quinol-4-yl) [(2S,4S,5R)-5-
pipeeolyl-L-arginine-4-nitroamlide dihydroehloride in water to give
vinylquinuclidin-2-yl]methanol;
a 0.003 M solution. Dilute before use in titrating in
C19H22N20 = 294.4 (485-71-2)
tris(hydroxymethyl)aminomethane-EDTA buffer solution pH 8.4
to give a 0.0005 M solution. White or almost white, crystalline powder, very slighdy
soluble in water and in light petroleum, soluble in ethanol
Chromogenic Substrate R3 Dissolve D-valyl-leueyl-lysyl-
(96%).
4-nitroanilzde dihydroehloride in water to give a 0.003M solution.
[a]g>, - 105 to -110, determined on a 5% w/v solution in
Chromogenic Substrate R4 Dissolve D-phenylalanyl-L-
ethanol (96%); melting point, about 208°, with
pipeeolyl-L -arginine-4-nitroanilide dihydroehloride in water to give
decomposition.
a 0.008M solution. Dilute to 0.0025 M with phosphate buffer
solmion pH 8.5 before use. Store protected from light.
Chromogenic Substrate R5 Dissolve N-benzoyl-L- Cinchonine (S)-(Quinol-4-yl) [(2R,4S,5R)-5-
isoleueyl-L-glutamyl-glyeyl-L-arginine-4-nitroanilide hydroehloride vinylquinuclidin-2-yl]methanol;
in water to give a 0.003M solution. C19H22N 20 = 294.4 (118-10-5)
Chromophore Substrate Rl See Chromogenie White or almost white, crystalline powder, very slighdy
substrate R1. soluble in water, sparingly soluble in ethanol (96%) and in
methanol.
Chromophore Substrate R2 See Chromogenie
substrate R2. [a]g>, +225 to +230, determined on a 5% w/v solution in
ethanol (96%); melting point, about 263°.
Chromophore Substrate R3 See Chromogenic
substrate R3. Store protected from light.
Chromotrope I1B Chromotrope 2B; Cl 16575; Cineole 1,8-Cineole, Eucalyptol, 1,8-epoxy-p-menthane;
C16H9N3Na201OS2 = 513.4 (548-80-1) C lOH 1SO = 154.3 (470-82-6)
Reddish-brown powder, soluble in water giving a yellowish- Colourless liquid, practically insoluble in water, miscible with
red colour, practically insoluble in ethanol (96%). anhydrous ethano!.
Chromotrope I1B Solution Chromotrope 2B solution d~g, 0.922 to 0.927; n~o, 1.465 to 1.459 .
A 0.005% w/v solution of ehromotrope IlB in sulfurie acid. Freezingpoint (2.2. 18): 0° to 1°.
Chromotropic Acid 4,5-Dihydroxy-2,7- Distillation range (2.2.11): 174° to 177°.
naphthalenedisulfonic acid; ClOHsOSS2 = 320.3 (148-25-4) PhenoZ Shake 1 g with 20 mL of water. Allow to separate
General reagent grade of commerce. and add to 10 mL of the aqueous layer 0.1 mL of jerric
ehloride solution R1. No violet colour develops.
Turpentine oiZ Dissolve 1 g in 5 mL of ethanol (90% v/v) .
Chromotropic Acid Sodium Salt Disodium 4,5-
Add dropwise freshly prepared bromine water. Not more than
dihydroxynaphthalene-2, 7-disulfonate; C lOH6Na20SS2,
0.5 mL is required to give a yellow colour lasting for
2H 20 = 400 .3 (5808-22-0)
30 minutes.
Residue on evaporation: maximum 0.05%.
V -A44 Appendix I A 2014
To 10.0 mL add 25 mL of water, evaporate on a water-bath Chromatography Thin-layer chromalOgraphy (2.2.27),

and dry the residue 10 constant mass at 100-105°. using si/ica gel GF254 as the coating substanee: apply 10 the
Cineole used in gas chromatography complies with the following plate 10 ¡.¡L of a 0.1 % w/v solution in toluene. Develop over a
additional test. path ofl5 em using a mixture of 15 volumes of ethyl acetate
and85 volumes of toluene. Allow the plate 10 dry in air
Assay Gas chromatography (2.2.28) as preseribed in the
andexamine in ultraviolet light at 254 nm.
monograph Peppermint oil (0405).
The chromalOgram shows only one principal spot.
Citral used in gas chromatography complies with the following
Content, minimum 98.0%, ca1culated by the normalisalÍon additional test.
procedure.
Assay Gas chromalOgraphy (2.2.28) as prescribed in the
1,4-Cineole 1-Methyl-4-( 1-methylethyl)-7- monograph Citronella oil (1609).
oxabicyc\o[2.2.1)heptane, 1-Isopropyl-4-methyl-7-
Content of citral (neral + geranial), minimum 95.0%,
oxabicyc\o[2.2.1)heptane; ClQH1SO = 154.3 (470-67-7)
calculated by the normalisation procedure.
Colourless liquid.
Citrate ButTered Saline
dio, about 0.900; nbo, about 1.445; boiling point, about
173°. Dissolve 11.76 g of sodium citrate in 1800 mL of water,
adjust to pH 7.0 with 1M hydrochloric acid and add suffient
Cinnamaldehyde Cinnamic aldehyde, 3-Phenylpropenal; water 10 produce 2000 mL.
C 9 H sO = 132.2 (104-55-2)
Citrated Rabbit Plasma
Yellowish or greenish-yellow, oily liquid, slight1y soluble in
water, very soluble in ethanol (96%). Colleet blood by intracardiac puncture from a rabbit kept
fasting for 12 hours, using a plastic syringe with a No. 1
dig, 1.04810 1.051; n~, about 1.620. needle containing a suitable volume of 3.8% w/v solution of
SlOre protected from light. sodium citrate so that the final volume ratio of citrate solution
Cinnamamide (E)-3-Phenylprop-2-enamide; to blood is 1:9. Separate the plasma by centrifugation at
C 9H 9NO = 147.2 (62 1-79-4) 1500 g 10 1800 g at 15° 10 20° for 30 minutes.
White or almost white powder. SlOre at 0° 10 6°.
Melting point, about 149°. Use within 4 hours of collection.
Cinnamic Acid C 9 H s0 2 = 148.2 (140-10-3) Citric Acid C 6H s0 7 ,H 20 = 210.1 (5949-29-1)
General reagent grade of commerce. Citric Acid Monohydrate of the British Pharmacopoeia.
Melting point, about 133°. When used in the limit test for iron, Appendix VII, complíes
trans-Cinnamic Acid trans-3-Phenylacrylie acid; (2E)-3- with the following additional requirement.
Phenylprop-2-enoic acid; C 9 H S0 2 = 148.2 (140-10-3) Dissolve 0.5 g in 10 mL of water, add 0.1 mL of thioglycollic
Colourless crystals, very slight1y soluble in water, freely acid R, mix and make alkaline with ammonia. Dilute 10
soluble in ethanol (96%). 20 mL with water. No pink colour appears in the solution.
Me\ting point, about 133°. Citric Acid, Anhydrous C 6H s0 7 = 192.1 (77-92-9)
Cinnamic Aldehyde See Cinnamaldehyde. Anhydrous Citric Acid of the British Pharmacopoeia.
trans-Cinnamic Aldehyde (E)-3-Phenylprop-2-enal; Citric-Molybdic Acid Solution Mix 54 g of
CgHsO = 132.2 (14371-10-9) molybdenum(vI) oxide with 200 mL of water, add 11 g of
trans-Cinnamic aldehyde used in gas chromatography complies sodium hydroxide and heat, with stirring, until almost
complete solution has been obtained. Dissolve 60 g of citric
with the following additional test.
acid in 250 mL of water and add 140 mL of hydrochloric acid.
Assay Examine by gas chromatography, Appendix III B, Add the first solution to the seeond, stirring eontinuously,
under the eonditions described in the test for cool, filter if necessary, dilute 10 1000 mL with water and
ChromalOgraphic profile in the monograph for Cassia Oil add, dropwise, sufficient of a 1% w/v solution of potassium
using the reagent being examined. bromate to discharge the green colour.
The content is not less than 99 .0%, ca1culated by SlOre in a well-c\osed container, protected from light.
normalisation.
Citronellal 3,7-Dimethyl-6-octenal;
Cinnamyl Acetate 3-Phenylprop-2-en-1-yl acetate; ClOH1 SO = 154.3 (106-23-0)
C l l H 120 2 = 176.2 (103-54-8)
Very slight1y soluble in water, soluble in ethanol (96%).
n~, about 1.542; boiling point, about 262°.
dig, 0.848 10 0.856; n~, about 1.446.
Cinnamyl acetate used in gas chromatography complies with the
following additional test. Citronellal used in gas chromatography complies with the following
additional test.
Assay Examine by gaschromatography, Appendix III B,
under the conditions described in the test for Assay Carry out the method described in the monograph
Chromatographic profile in the monograph for Cassia Oil for Citronella Oil. The content is not less than 95.0%
using the reagent being examined. ca1culated by the normalisation procedure.
The content is not less than 99.0%, ca1culated by Citronello1 3,7-Dimethyloct-6-en-1-01;
normalisation. C lO H 20 0 = 156.3 (106-22-9)
Citral 3,7-Dimethylocta-2,6-dienal, Mixture of (2E)- and Clear, colourless liquid, practically insoluble in water,
(2Z)-3,7-Dimethylocta-2,6-dienal; miscible with ethanol (96%).
0
ClQH 160 = 152.2 (5392-40-5) dig, 0.857; nbo , 1.456; boiling point, 220 to 222°.
Light yellow liquid, practically insoluble in water, miscible Citronellol used in gas chromatography complies with the following
with ethanol (96%) and with propylene glycol. additional test.
Assay Carry out the method described in the monograph 1 volume in 10 (equivalent to 100% offactor V), 1 volume
for Citronella Oil. The content is not less than 95.0% in 50 (20%),1 volume in 100 (10%), and 1 volume in 1000
calculated by the nomzalisation procedure. (1 %). Using two-way logarithmic paper plot the average
Store in an airtight container, protected from light. coagulation times for each dilution of human plasma against
the equivalent percentage of factor V and read the percentage
Citronellyl Acetate 3,7 -Dimethyl-6-octen-l-yl acetate;
of factor V for the two dilutions of the factor V solution by
C 12H 22 0 2 = 198.3 (150-84-5)
interpolation. The mean of the two results gives the
d~g, 0.890; n~, 1.443; boiling point, 229°. percentage of factor V in the solution to be examined.
Citronellyl acetate used in gas chromatography complies with the Store in the frozen state at a temperature not higher
following additional test. than - 20°.
Assay Carry out the method described in the monograph Cobalt(u) Acetate Cobaltous acetate;
for Citronella Oil. The content is not less than 97.0% (CH3C02)2CO,4H20 = 249.1 (6147-53-1)
calculated by the nomzalisation procedure.
Store in an airtight container, protected from light.
Cobalt Ch10ride See Cobalt(lI) ehloride.
Citropten 5,7 -Dimethoxy-2H-1-benzopyran-2-one,
Cobalt(n) Ch10ride Cobalt chloride; cobaltous chloride;
5,7 -Dimethoxycoumarin, Limettin;
CoCI 2,6H 20 = 23.7.9 (7791-13-1)
C ll H lO 0 4 = 206.2 (487-06-9)
Red, crystalline powder or deep-red crystals, very soluble in
Needle-shaped crystals, practically insoluble in water and in
water, soluble in ethanol (96%).
light petroleum, freely soluble in acetone and in ethanol
(96%). Cobalt Nitrate See Cobalt(¡¡) nitrate.
Melting point, about 145°. Cobalt(u) Nitrate Cobalt Nitrate, Cobaltous nitra te;
Co(N0 3)z,6H 20 = 291.0 (10026-22-9)
Homogeneity Carry out the method for thin-layer
chromatography, Appendix III A, using silica gel GF254 as the Small garnet-red ctystals, very soluble in water.
coating substance and a mixture of 15 volumes of ethyl Codeine ClsH21N03,H20 = 317.4 (76-57-3)
acetate and 85 volumes of toluene as the mobile phase. Apply Of the British Pharmacopoeia.
to the plate 10 ¡.tL of a 0.01% w/v solution of the reagent Codeine Phosphate
being examined in toluene. After removal of the plate, allow it ClsH21N03,H3P04,v2H20 = 406.4 (41444-62-6)
to dry in air and examine under ultraviolet light (254 nm).
The chromatogram obtained shows only one principal spot. Codeine Phosphate Hemihydrate of the British
Pharmacopoeia.
Clobetasol Propionate 21-Chloro-9-fiuoro-ll~, 1.7-
2,4,6-Collidine 2,4,6-Trimethylpyridine;
dihydroxy-16~-methylpregna-1,4-diene- 3,20-dione
17-propionate; C2sH32ClFOs = 46.7.0 (25122-46-7) CsHIlN = 121.2 (108-75-8)
White or almost white crystalline powder, insoluble in water, General reagent grade of commerce.
soluble in ethanol (96%) and in acetone. A colourless to pale yellow liquid; boiling point, about 1.70°;
weight per mL, about 0.92 g.
[Ctl~O, about +104 (in dioxan); melting point, about 196°.
It may be stabilised by the addition of aluminium oxide.
Coagulation Factor V Solntion Clotting factor V
solution Congo Red Cl 22120; Disodium (biphenyl-4,4'-diyl-bis-
2,2' -azo) bis( 1-aminonaphthalene-4-sulfonate);
Coagulation factor V solution may be prepared by the
following method or by any other method which excludes C32H22NóNa20óS2 = 697 (573-58-0)
factor VIII. Brownish-red powder, soluble in water.
Prepare the factor V reagent from fresh oxalated bovine Congo Red Fibrin Take 1.5 g of fibrin and leave
plasma, by fractionation at 4° with a saturated solution of overnight in 50 mL of a 2.0% w/v solution of congo red in
ammonium sulfate prepared at 4°. Separate the fraction which ethanol (90% v/v). Filter, rinse the fibrin with water and store
precipitates between 38% and 50% of saturation, which under ether.
contains factor V without significant contamination with Congo Red Paper lmmerse strips of filter paper for a few
factor VIII. Remove the ammonium sulfate by dialysis and minutes in congo red solution. AlIow to dry.
dilute the solution with a 0.9% w/v solution of sodium chloride Congo Red Solntion Dissolve 0.1 g of congo red in a
to give a solution containing between 10% and 20% of the mixture of 20 mL of ethanol (96%) and water and dilute to
quantity of factor V present in fresh human normal plasma. 100 mL with water.
Assay offactor V Prepare two dilutions of the preparation Sensitivity To 0.2 mL of the congo red solution add
of factor V in imidazole buffer solution pH 7.3 containing 100 mL of carbon dioxide-free water and 0.3 mL of 0.1M
1 volume of the preparation in 10 volumes and in hydrochloric aeid. The solution is blue. Not more than 0.3 mL
20 volumes of the buffer solution respectively. Test each of 0.1M sodium hydroxide is required to change the colour to
dilution as follows: mix 0.1 mL of plasma substrate deficient in pink.
factor V, 0.1 mL of the solution to be examined, 0.1 mL of Colour change pH 3.0 (blue) to pH 5.0 (pink) .
thromboplastin and 0.1 mL of a 0.35% w/v solution of calcium
ehloride and measure the coagulation times, Í.e. the interval Coomassie BIne See Acid Blue 92.
between the moment at which the calcium chloride solution Coomassie BIne Solntion See Acid Blue 92 solution.
is added and the first indication of the formation of fibrin, Coomassie Staining Solntion A 0.125% w/v solution of
which may be observed visually or by means of a suitable aeid blue 83 in a mixture consisting of 1 volume of glacial
apparatus. acetie acid, 4 volumes of methanol and 5 volumes of water.
In the same manner, determine the coagulation time (in Filter.
duplicate) of four dilutions of human normal plasma in Coomassie Staining Solntion Rl Dissolve 0.2.75 g of
imidazole buffer solution pH 7.3, containing respectively, brilliant blue R in 200 mL of methanol. Stir until complete
dissolution of the crystals (for about 2 hours). Add 750 mL through a sintered-glass filter (40) (2.1.2), wash with water
of water and 50 mL of glacial acetic acid. Stir ovemight (for until the filtra te is c1ear and take up the precipitate with
at least 16 hours); filter. 200 mL of concentrated ammonia. Filter through a sintered-
Copper Cu = 63.55 (7440-50-8) glass filter (2.1.2) and repeat the filtration to reduce the
residue to a minimum.
Cleaned foil, tumings, wire or powder of the pure metal of
e1ectrolytic grade. Corallin CI 43811; sodium salt ofrosolic acid (603-45-2)
Copper Acetate See Copper(lI) acetate. General reagent grade of commerce.
Copper(n) Acetate Copper Acetate, Cupric aceta te; Hard, dull red masses.
C4H 6CU04,H20 = 199.7 (142-71-2) Corallin Solution, Alkaline Dissolve 5 g of corallin in
Blue-green crystals or powder, freely soluble in boiling water, 100 mL of ethanol (90%). Immediately before use add 1 mL
soluble in water and in ethanol (96%), slight1y soluble in of the solution to 20 mL of a 20% w/v solution of sodium
glycerol (85%). carbonate.
Copper Carbonate Approximately CuC0 3 ,Cu(OH)z,H zO Cortisone C21H2S0S = 360.4 (53-06-5)
(12069-69-1) Content, minimum 95.0% .
General reagent grade of commerce. Me1ting point, 223 0 to 228°.
Copper(n) Chloride Cupric chloride; Cortisone Acetate CZ3H300 6 = 402.5 (50-04-4)
CuCI 2,2H zO = 170.5 (10125-13-0) Of the British Pharrnacopoeia.
Greenish-blue powder or crystals, deliquescent in moist air, Coumaphos Cl4Hl6ClOsPS = 362.8 (56-72-4)
efflorescent in dry air, free1y soluble in water, in ethanol Melting point, 91 0 to 92°.
(96%) and in methanol, sparingly soluble in acetone .
A suitable certified reference solution (10 ng/¡.¡L in iso-
octane) may be used.
Copper Chloride-Pyridine Reagent Dissolve 40 mg of o-Coumaric Acid (E)-2-Hydroxycinnamic acid; (2E)-3-
copper(ll) chloride in pyridine, warming until complete (2-Hydroxyphenyl)prop-2-enoic acid;
dissolution is effected, and coo!. Add 1 mL of carbon disulfide C 9 H s 0 3 = 164.2 (614-60-80)
and sufficient pyridine to produce 100 mL.
White or almost white powder.
Copper Edetate Solution To 2 mL of a 2% w/v solution
of copper(ll) acetate add 2 mL of O.lM disodiwn edetate and Melting point, about 217 0 •
dilute to 50 mL with water. p-Coumaric Acid 4-Hydroxycinnamic acid; 3-
Copper Nitrate See Copper(ll) nitrate. (4-Hydroxyphenyl)-prop-2-enoic acid;
C9H s 0 3 = 164.2 (7400-08-0)
Copper(n) Nitrate Chloride dinitrate trihydrate, cupric
nitra te, copper nitrate; White or almost white needles, practically insoluble in water,
Cu(N0 3 )z,3HzO = 24l.6 (10031-43-3) soluble in acetone and in methano!.
0
Dark blue crystals, hygroscopic, very soluble in water giving a Me1ting point, 214 to 217".
strongly acid reaction, freely soluble in ethanol (96%) and in p-Coumaric acid used in the assay in Neule leaf (1897) complies
dilute nitric acid. with the following additional tests.
Store in an airtight container. Loss on drying (2.2.32) Maximum 5.0%, deterrnined on
Copper Oxide Solution, Arnmoniaca1 Triturate 0.5 g of 0.200 g by drying in an oven at 105 0 for 2 hours .
copper carbonate with 10 mL of water and gradually add Assay Liquid chromatography (2.2.29) as prescribed in the
10 mL of 13.5M ammonia. monograph on Nettle leaf.
Copper Sulfate See Copper(lI) sulfate. Content Minimum 95%, ca1culated by the normalisation
Copper(n) Sulfate Copper sulfate, copper sulphate, procedure.
copper(,,) sulphate, cupric sulfate, cupric sulphate; Coumarin 2H-Chromen-2-one, 2H-1-Benzopyran-2-one;
CuS04, 5H zO = 249.7 (7758-99-8) C 9 H 60 2 = 146.1 (91-64-5)
Blue powder or deep-blue crystals, slowly efflorescent, very Colourless, crystalline powder or orthorhombic or rectangular
soluble in water, slightly soluble in ethanol (96%). crystals, very soluble in boiling water, soluble in ethanol
Copper Sulfate-Pyridine Reagent Copper sulphate- (96%). It dissolves in solutions of alkali hydroxides.
pyridine reagent Melting point, 68 0 to 70°.
Dissolve 4 g of copper(lI) sulfate in 90 mL of water and add Coumarin used in gas chromatography complies with ¡he following
30 mL of pyridine. additional test.
Prepare immediately before use. Assay Gas cmomatography (2.2.28) as prescribed in the
Copper Sulfate Solution Copper sulphate solution monograph Cassia oi! (1496).
A 12.5% w/v solution of copper(lI) sulfate. Content, minimum 98.0%, ca1culated by the normalisation
procedure.
Copper Sulfate Solution, Weak Copper sulphate
solution, weak Coumestro1 C1sHsOs = 268.22 (479-13-0)
A 10% w/v solution of copper(lI) sulfate. General reagent grade of commerce.
Copper Tetramrnine, Arnmoniacal Solution of Creso1 See o-Creso!.
Dissolve 34.5 g of copper sulfate in 100 mL of water and, m-Cresol Metacresol of the British Pharrnacopoeia.
whilst stirring, add dropwise concentrated ammonia until the o-Cresol Cresol, 4-Methylphenol;
precipitate which forms dissolves completely. Keeping the C 7 H s O = 108.1 (95-48-7)
temperature below 20°, add dropwise with continuous Crystals or a super-cooled liquid becoming dark on exposure
shaking 30 mL of strong sodium hydroxide solution. Filter to Iight and air, miscible with anhydrous ethanol, soluble in
2014 Appendix 1 A V -A4 7
about 50 parts of water and soluble in solutions of alkali small quantities. Titrate with O.l M sodium thiosulfate VS using
hydroxides. 0.5 mL of starch solution, added towards the end of the
dig, about 1.05; nbo, 1.540 to 1.550; boiling point, about titration, as indicator.
190°. 24.5 to 25.5 mL of O.lM sodium thiosulfate VS is used in the
Freezing point (2.2.18): minimum 30S. titration.
Residue on evaporation: maximum 0.1 % mlm, B. Dilute 10.0 mL to 100.0 mL with water and mix.
determined by evaporating on a water-bath and drying in an To 10.0 mL ofthe solution, add 25.0 mL of
oven at 100-105°. O.IM hydrochloric acid VS and heat for 1 hour on a water-
Store protected from light, moisture and oxygen. bath. Cool, adjust with water to the initial volume and titrate
with O.IM sodium hydroxide VS, using 0.1 mL of
Distil before use. phenolphthalein solution R1 as indicator.
p-Cresol 4-Methylphenol; C 7H sO = 108.1 (106-44-5) 5.7 mL to 6.3 mL of O.IM sodium hydroxide VS is used in the
Colourless or white or almost white crystals or crystalline titration.
mass. C. Dilute 10.0 mL to 100.0 mL with water and mix. Titrate
dig, about 1.02; boiling point, about 202°. 10.0 mL ofthe solution with O.IM hydrochloric acid VS, using
m-Cresol Purple m-Cresolsulfonphthalein; 0.1 mL of phenolphthalein solution R1 as indicator.
C21H1SOSS = 382.4 (2303-01-7) 6. O mL to 7.5 mL of 0.1 M hydrochloric acid VS is used in the
Olive-green, crystalline powder, slightly soluble in water, titration.
soluble in ethanol (96%), in glacial acetic acid and in Cupriethylenediamine hydroxide solution (14552-35-3)
methanol. The molar ratio of ethylenediamine to copper is
m-Cresol Purple Solution Dissolve 0.1 g of m-cresol 2.00 ± 0.04.
purple in 13 mL of O.OlM sodium hydroxide, dilute to 100 mL This solution is commercially available.
with water and mix.
Cupri-tartaric Solution
Colour change pH 1.2 (red) to pH 2.8 (yellow); pH 7.4
(yellow) to pH 9.0 (purple). Solution A Dissolve 34.6 g of copper(lI) sulfate in water and
dilute to 500 mL with the same solvent.
Cresol Red 4,4'-(3H-2, 1-Benzoxathiol-3-ylidene)di-o-
cresol S,S-dioxide; Cresolsulfonphthalein; Solution B Dissolve 173 g of potassium sodium (+) -tartrate
C21H1SOSS = 382.4 (1733-12-6) and 50 g of sodium hydroxide in 400 mL of water. Heat to
boiling, allow to cool and dilute to 500 mL with carbon
A reddish-brown crystalline powder, slightly soluble in water, dioxide-free water.
soluble in ethanol (96%) and in dilute solutions of alkali
hydroxides. Mix equal volumes of the 2 solutions immediately before use.
Cresol Red Solution Dissolve 0.1 g of cresol red in a Cupri-tartaric Solution Rl Fehling's solution
mixture of 2.65 mL of 0.1 M sodium hydroxide and 20 mL of Solution A Dissolve 34.6 g of copper(lI) sulfate in a mixture
ethanol (96%) and dilute to 100 mL with water. of 0.5 mL of sulfun'c acid and sufficient water to produce
Testfor sensitivity A mixture of 0.1 mL of the cresol red 500 mL.
solution and 100 mL of carbon dioxide-free water to which Solution B Dissolve 176 g of potassiwn sodium (+) -tartrate
0.15 mL of 0.02 M sodium hydroxide has been added is and 77 g of sodium hydroxide in sufficient water to produce
purple-red. Not more than 0.15 mL of 0.02 M hydrochloric 500 mL.
acid is required to change the colour to yellow. Mix equal volumes of solutions A and B immediately before
Colour change pH 7.0 (yellow) to pH 8.6 (red). use.
CrystaI Violet Cl 42555; basic violet 3; Cupri-tartaric Solution R2 Dilute potassium
C2sH30CIN3 = 408.0 (548-62-9) cupri-tartrate solution
Dark-green powder or crystals, soluble in water and in Add 1 mL of a solution containing 0.5% w/v of copper(lI)
ethanol (96%). sulfate and 1% w/v of dipotassium (+) -tartrate to 50 mL of
CrystaI Violet Solution Dissolve 0.5 g of crystal violet in sodium carbonate solution R1 . Prepare irnmediately before use.
anhydrous acetic acid and dilute to 100 mL with the same Cupri-tartaric Solution R3 Prepare a solution containing
solvent. 1.0% w/v of copper sulfate and 2.0% w/v of sodium tartrate.
Testfor sensitivity To 50 mL of anhydrous acetic acid add To 1.0 mL of the solution add 50 mL of sodium carbonate
0.1 mL ofthe crystal violet solution. On addition ofO .l mL solution R2. Prepare immediately before use.
of 0.1 M perchloric acid the bluish-purple solution turns Cupri-tartaric Solution R4
bluish-green. Solution A 15.0% w/v copper(lI) sulfate.
Cupric Chloride See Copper(lI) chloride. Solution B Dissolve 2.5 g of anhydrous sodium carbonate,
Cupri-citric Solution Dissolve 25 g of copper(lI) sulfate, 2.5 g of potassium sodiwn (+)-tartrate, 2.0 g of sodiwn
50 g of citric acid and 144 g of anhydrous sodiuJ'n carbonate in hydrogen carbonate, and 20.0 g of anhydrous sodium sulfate in
water and dilute to 1000 mL with the same solvent. water and dilute to 100 mL with the same solvent.
Cupri-citric Solution Rl Dissolve 25 g of copper(n) Mix 1 part of solution A with 25 parts of solution B
sulfate, 50 g of cim'c acid and 144 g of anhydrous sodium immediately before use.
carbonate in water and dilute to 1000 mL with the same Curcurnin 1,7-bis(4-Hydroxy-3-methoxyphenyl)hepta-l ,6-
solvent. diene-3,5-dione; C21H2006 = 368.4 (458-37-7)
Adjust the solution so that it complies with the following Orange-brown, crystalline powder, practically insoluble in
requirements. water, soluble in glacial acetic acid.
A. To 25 .0 mL add 3 g of potassium iodide. Add 25 mL of a Melting point, about 183 8
•
25% w/w solution of sulfuric acid with precaution and in
Curcurninoids A mixture of curcumin (C21H2006 Cyclohexylenedinitrilotetra-acetic Acid (± )-trans-l ,2-

= 368.4), demethoxycurcumin (C2oH1SOS = 338.4) and Diaminocyclohexane-N,N,N' ,N' -tetra-acetic acid;
biS-demethoxycurcumin (C19H1604 = 308.3) C14H22N20S,H20 = 364.4 (13291-61-7)
Cyanoacetic Acid Malonic acid mononitrile; White or almost white, crystalline powder.
C 3H 3 N0 2 = 85.1 (372-09-8) Melting point, about 204°.
White or yellowish-white, hygroscopic crystals, very soluble in Cyclohexylmethanol Cyclohexylcarbinol;
water. C 7 H 14 0 = 114.2 (100-49-2)
Srore in an airtight container. Liquid with a slight odour of camphor, soluble in ethanol
Cyanocobalarnin COIX-[IX-(5,6-Dimethylbenzimidazolyl)]- (96%).
Cop-cyanocobamide; vitamin B 12; nfi, about 1.464; boiling point, about 185°.
C63HssCoN14014P = 1355 (68-19-9) 3-Cyclohexylpropionic Acid
Of the British Pharmacopoeia. C 9H 16 0 2 = 156.2 (701-97-3)
Cyanogen Bromide Solution Add dropwise, with cooling Clear liquid.
O.IM ammonium thiocyanate to bromine water until the yellow
d~g, about 0.998; n~, about 1.4648; boiling point, about
colour disappears . Prepare immediately before use. 130°.
Cyanoguanidine See 1-Cyanoguanidine.
Cyhalothrin C23H19CIF3N03 = 449.9 (91465-08-6)
l-Cyanoguanidine Cyanoguanidine, Dicyandiamide;
Boiling point, 187° ro 190°; melting point, about 49°.
C 2H 4 N 4 = 84.1 (461-58-5)
A suitable certified reference solution (1 O ng/~L in
White or almost white, crystalline powder, sparingly soluble cyclohexane) may be used.
in water and in ethanol (96%), practically insoluble in
methylene chloride. p-Cymene l-Isopropyl-4-methylbenzene,
4-Isopropyltoluene; C lO H 14 = 134.2 (99-87-6)
Melting point, about 21 O°.
Colourless liquid, practically insoluble in water, soluble in
Cyclobutane-l,l-dicarboxylic Acid C 6H lO 0 4 = 144.1
ethanol (96%).
(5445-51-2)
d~g, about 0.858; n~o, about 1.4895; boiling point, 175° to
178°.
p-Cymene used in gas chromatography complies with the following
ex-Cyclodextrin Cyclohexakis-(1-->4)-(ex-n-glucopyranosyl), additional test.
Cyclomaltohexaose, Alfadex; C36H60030 = 972 (10016-20-3) Assay Gas chromatography (2.2.28) as prescribed in the
~-Cyclodextrin Betadex of the British Pharmacopoeia. monograph Peppermint oil (0405) .
y-Cyclodextrin Gamma-cyclodextrin; Test solution The substance to be examined.
C4sH so040 = 1297 (17465-86-0)
Content, minimum 96.0%, calculated by the normalisation
General reagent grade of commerce. procedure.
~-Cyclodextrin for Chiral Chromatography, Modified Cynarin (IR,3R,4S,5R)-1,3-Bis[[3-(3,4-
30% of 2,3-di-O-ethyl-6-0-tert-butyldimethylsilyl-~ dihydroxyphenyl)propenoyl]oxy]-4,5-
cyclodextrin dissolved in dihydroxycyclohexanecarboxylic acid;
poly(dimethyl) (85) ( diphenyl) (15) siloxane. C2sH2401 2 = 516.4 (30964-13-7)
~-Cyclodextrin for Chiral Chromatography, White or almost white amorphous mass, odourless.
Modified Rl Cypermethrin C22H19ClzN03 = 416.3 (52315-07-8)
30% of 2,3-di-O-acetyl-6-0-tert-butylsilyl-~- cyclodextrin Boiling point, 170° to 195°; melting point, 60° ro 80°.
dissolved in poly(dimethyl) (85)(diphenyl) (15)siloxane.
A suitable certified reference solution (1 O ng/~L in
Cyclohexane C 6H 12 = 84.2 (110-82-7) cyclohexane) may be used.
Clear, colourless, fiammable liquid, practically insoluble in L-Cysteine C 3H 7N0 2S = 121.2 (52-90-4)
water, miscible with organic solvents.
Powder, freely soluble in water, in ethanol (96%) and in
d~g, about 0.78; boiling point, about 80S;. acetic acid, practically insoluble in acetone.
Cyclohexane used in spectrophotometry complies with the following Cysteine Hydrochloride
additional test. C 3H 7 N0 2S,HCI,H 20 = 175.6 (7048-04-6)
Transmittance Not less than 45% at 220 nm, 70% at Cysteine Hydrochloride Monohydrate of the British
235 nm, 90% at 240 nm and 98% at 250 nm, using water in Pharmacopoeia.
the reference cel!.
L-Cystine C6H1 2N204S2 = 240.3 (56-89-3)
Cyclohexane Rl Complies with the requirements
White or almost white, crystalline powder, practically
prescribed for cyclohexane with the following additional
insoluble in water and in ethanol (96%). It dissolves in dilute
requirement.
solutions of alkali hydroxides.
The fiuorescence, me~sured at 460 nm, under illumination
[Q'l~o, - 218 to -224, determined in 1M hydrochloric acid.
with an excitant light beam at 365 nm, is not more intense
than that of a solution containing 0.002 ppm of quinine in Melting point, 250°, with decomposition.
0.05 M sulfuric acid. Cytosine C 4H sN 30 = 111.1 (71-30-7)
Cyclohexylarnine C6H13N = 99 .2 (108-91-8) Content, minimum 95.0%.
Colourless liquid, soluble in water, miscible with usual Daidzein 7 -Hydroxy-3-(4-hydroxyphenyl)-4H-l-
organic solvents. benzopyran-4-one; C 1sH lO 0 4 = 254.2 (486-66-8)
n~, about 1.46; boiling point, 134° to 135°.
Daidzin Daidzein-7 -O-glucoside, 7-(~-D Colourless needles, soluble in dimethyl sulfoxide and in hot
Glucopyranosyloxy)-3-(4-hydroxyphenyl)-4H-1-benzopyran- methano!.
4-one; C21H2009 = 416 .4 (552-66-9) Melting point, about 288°.
Dantron See 1,8-Dihydroxyanthraquinone. 2-DeoXY-D-ribose Thyminose. 2-DeoXY-D-erythro-
o,p'-DDD 1-(2-Chlorophenyl)-1-(4-chlorophenyl)-2,2- pentose; C 5H lO 0 4 = 134.1; (533-67-5)
dichloroethane; C l4H lO Cl 4 = 320.0 (53-19-0) 2' -Deoxyuridine 1-(2-Deoxy-~-d-erythro-pentofuranosyl)
A suitable certified reference solution (10 ng/I1L in lH,3H-pyrimidine-2,4-dione; C9HIZNz05 = 228 .2
cyclohexane) may be used. (951-78-0)
p,p' -DDD 1, l-bis(4-Chlorophenyl)-2,2-dichloroethane; Melting point, about 165 0 •
C l4H lO Cl4 = 320.0 (72-54-8) Chromatography Thin-Iayer chromatography (2. 2.27) as
Boiling point, about 193°; melting point, about 109°. prescribed in the monograph Idoxuridine (0669): apply 5 I1L
A suitable certified reference solution (10 ng/¡tL in of a 0.025% w/v solution ; the chromatogram shows only
cyclohexane) may be used. one principal spot.
o,p '-DDE 1-(2-Chlorophenyl)-l-(4-chlorophenyl)-2,2- 4-Desoxypyridoxine Hydrochloride 5-(Hydroxymethyl)-
dichloroethylene; C l4H s Cl 4 = 318.0 (3424-82-6) 2,4-dimethylpyridin-3-01; C sH 12N0 2CI = 189.6 (148-51-6)
A suitable certified reference solution (10 ng/I1L in Destaining Solution A mixture of 1 volume of glacial
cyclohexane) may be used. acetic acid, 4 volumes of methanol and 5 volumes of water.
p,p'- DDE 1, 1-Bis(4-Chlorophenyl)-2,2-dichloroethylene; Deuterated Acetic Acid C 2D 40 2 = 64.1 (1186-52-3)
C l4H s C4 = 318.0 (72-55-9) Degree of deuteration, mínimum 99.7%.
Boiling point, 316° to 317°; melting point, 88° to 89°. dig, about 1.12; ni?, about 1.368; boiling point, about 115°;
A suitable certified reference solution (10 ng/I1L in melting point, about 16°.
cyclohexane) may be used. Deuterated Acetone Deuteroacetone; C 3D 6 0 = 64.1
o,p'-DDT 1-(2-Chlorophenyl)-1-(4-chlorophenyl)-2,2,2- (666-52-4)
trichloroethane; C l4 H 9Cl5 = 354.5 (789-02-6) Degree of deuteration, mínimum 99.5% .
A suitable certified reference solution (10 ng/I1L in Clear, colourless liquid, miscible with water, with
cyclohexane) may be used. dimethylformamide, with anhydrous ethanol and with
p,p ' - DDT 1, 1-bis(4-Chlorophenyl)-2,2,2-trichloroethane; methano!.
C l4H 9Cl5 = 354.5 (50-29-3) dig, about 0.87; ni?, about 1.357; boiling point, about 55°.
Boiling point, about 260°; melting point, 108° 10 109°. Water and deuterium oxide Not more than 0.1 %.
A suitable certified reference solution (10 ng/I1L in Deuterated Acetonitrile C 22H 3N = 44.1 (2206-26-0)
cyclohexane) may be used. Degree of deuteration, mínimum 99.8%.
Decana! Decyl aldehyde; C lO H 20 0 = 156.3 (112-31-2) Clear, colourless liquid, miscible with water, with acetone
Oily, colourless liquid, practically insoluble in water. and with methano!.
Decanal used in gas chromatography complies with the following dig, about 0.78; ni?, about 1.344.
additional test. Deuterated Chloroform See Deuterochloroform.
Assay Gas chromatography (2.2.28) as prescribed in the Deuterated Dimethyl Sulfoxide Deuterated dimethyl
monograph Sweet orange oil. sulphoxide, CZH 6)-dimethyl sulfoxide, CZH 6)-dimethyl
Content, minimum 97%, calculated by the normalisation sulphoxide, dimethyl sulfoxide-d6 , dimethyl sulphoxide-d6 ;
procedure. C 2D 60S = 84.2 (2206-27-1)
Decane See n-Decane. Degree of deuteration, mínimum 99 .8%.
n-Decane C IO H 22 = 142.3 (124-18-5) Very hygroscopic liquid, practically colourless, viscous,
Colourless liquid, practically insoluble in water. soluble in water, in acetone and in anhydrous ethano!.
nbo, about 1.411; boiling point, about 174°. dig, about 1.18; melting point, about 20°.
Decanol See Decan-1-ol. Water and deuterium oxide Maximum 0.1% .
Decan-l-ol Decanol, n-decyl alcohol; C lO H 22 0 = 158.3 Store in an airtight container.
(112-30-1) Deuterated Methanol CZH)-Methanol; methanol-d;
Viscous liquid, solidifying at about 6°, practically insoluble in CD 40 = 36.1 (811-98-3)
water, soluble in ethanol (96%). Degree of deuteration, mínimum 99 .8% .
ni?, about 1.436; boiling point, about 230°. Clear, colourless liquid miscible with water, with ethanol
Deltamethrin C22HI9Br2N03 = 505 .2 (52918-63-5) (96%) and with methylene chloride.
Boiling point, about 300°; melting I?oint, about 98°. dig, about 0.888; ni?, about 1.326; boiling point, 65.4°.
A suitable certified reference solution (10 ng/I1L in Deuterated Sodium Trimethylsilylpropionate Sodium
cyclohexane) may be used. 3-(trimethylsilyl) (2,2,3,3-D 4 )propionate, TSP-d 4;
Demeclocycline Hydrochloride Of the British C6H9D4Na02Si = 172.3 (24493-21-8)
Pharmacopoeia. Degree of deuteration, mínimum 98%.
DemethyIflumazenil Ethyl 8-ftuoro-6-oxo-5,6-dihydro- White or almost white powder.
4H-imidazo [1 ,5-a] [1 A] benzodiazepíne-3-carboxylate; Deuterium Chloride Deuterated hydrochloric acid;
Cl4Hl2FN303 = 289.3 (79089-72-8) DCI = 37.47 (7698-05-7)
Gas.
V-ASO Appendix 1 A 2014
Degree of deuteration, minimum 99 %. White or almost white crystals or granules, hygroscopic, very
Caution Toxic. soluble in water, practically insoluble in ethanol (96%) .
Deuterium chIoride soludon pH (2.2.3) About 8 for a 200 gIL solution.
Dilute 1 mL of deuterium chloride (38% mlm) with 5 mL of Diatomaceous Earth See Diatomaceous suppon.
deuten'um oxide. Diatomaceous Earth for Gas Chromatography
Deuterium Oxide Deuterated water; DzO = 20.03 See Acid-washed diatomaceous support.
(7789-20-0) Diatomaceous Earth for Gas Chromatography Rl
Degree of deuteration, minimum 99.7%. White or almost white, fine granular powder, made up of
siliceous frustules of fossil diatoms or of debris of fossil
dig, about l.11; n~o, about 1.328; boiling point, about 101°. diatoms, practically insoluble in water and in ethanol (96%).
Deuterium Oxide Rl See Isotopically Pure Deuterium The substance may be identified by microscopic examination
Oxide with a magnification of x 500. The substance is purified by
Deuterium Oxide, Isotopically Pure Deuterium treating with hydrochloric acid and washing with water.
oxide R1 Particle size Maximum 5% is retained on a sieve No.
Deuterated water. 250. Maximum 10% passes a sieve No. 180.
Degree of deuteration, minimum 99.95% . Diatomaceous Earth for Gas Chromatography RZ
DeuterochIoroform e H)-Chloroform, chloroform-d; White or almost white, fine granular powder with a specific
CDCl 3 = 120.4 (865-49-6) surface area of about 0.5 mZ/g, made up of siliceous frustules
Degree of deuteration, minimum 99.7%. of fossil diatoms or of debris of fossil diatoms, practically
insoluble in water and in ethanol (96%). The substance may
Clear, colourless liquid, practically insoluble in water,
be identified by microscopic examination with a
miscible with acetone and with ethanol (96%). It may be
magnification of x 500. The substance is purified by
stabilised over silver foil.
treating with hydrochloric acid and washing with water.
dig, about 1.51; n~, about 1.445; boiling point, about 60°. Particle size Maximum 5% is retained on a sieve No.
Water and deuterium oxide Maximum 0.05%. 180. Maximum 10% passes a sieve No. 125.
Devarda's Alloy (8049-11-4) Copper, 50 parts; Diatomaceous Earth for Gas Chromatography,
aluminium, 45 parts; zinc, 5 parts. Silanised See Silanised diatomaceous support.
General reagent grade of commerce containing not more DiatOInaceous Earth for Gas Chromatography Rl,
than 20 ppm of nitro gen as NH4. Silanised Prepared from crushed pink firebrick and
Deve10per Solution Dilute 2.5 mL of a 2.0% w/v solution silanised with dimethyldichlorosilane or other suitable
of citric acid and 0.27 mL of formaldehyde to 500 mL with silanising agents. The substance is purified by treating with
water. hydrochloric acid and washing with water.
Dexamethasone C 22H z9FO s = 392.5 (50-02-2) Diatomaceous Filter-aid, Washed, F1ux-ca1cined To
General reagent grade of commerce. 500 g of fiux-ca1cined, diatomaceous filter-aid (Celite 545 is
Melting point, about 263°. suitable), add 2000 mL of hydrochloric acid, mix, allow to
stand with occasional stirring for 12 hours, filter and wash
Dextran for Chromatography RZ, Cross-linked Bead-
the residue with water until the washings are neutral to litmus
form dextran with a fraction range suitable for the separation
papel'. Continue washing the residue on the filter paper,
of peptides and proteins with relative molecular masses of 15
using 500 mL of methanol followed by 1000 mL of a mixture
x lO z to 30 x 103 . When dry, the beads have a diameter
of equal volumes of methanol and ether. Finally dry the
of 20 flm to 80 flm.
washed residue at 100° until the odour of solvent is no
Dextran for Chromatography R3, Cross-linked Bead- longer detectable. It should be stored in an airtight
form dextran with a fraction range suitable for the separation container.
of peptides and proteins with relative molecular masses of 4
Diatomaceous Support Diatomaceous earth (91053-39-3)
x 103 to 15 x 104. When dry, the beads have a diameter
of 40 flill to 120 flm. White or almost white, fine granular powder, made up of
siliceous frusrules of fossil diatoms or of debris of fossil
Dextrose See v-Glucose.
diatoms, practically insoluble in water and in ethanol (96%).
3,3' - Diaminobenzidine TetrahydrochIoride
The substance may be identified by microscopic examination
C¡zH¡-tN'4,4HCl,2HzO = 396.1 (7411-49-6)
with a magnification of x 500.
Almost white or slightly pink powder, soluble in water.
Diatomaceous Support, Acid-washed Diatomaceous
Melting point, about 280°, with decomposition. earth for gas chromatography
Diammonium 2,2 '-azinobis(3-ethy1benzothiazoline-6- White or almost white, fine granular powder, made up of
sulfonate) . ABTS, Diarnmonium siliceous frusrules of fossil diatoms or of debris of fossil
2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate), diatoms, practically insoluble in water and in ethanol (96%).
Diarnmonium 2,2'-(diazanediylidene)bis [3-ethyl-2,3- The substance may be identified by microscopic examination
dihydrobenzothiazole-6-sulfonate); C¡ SHZ-tN'60 6S4 = 548.7 with a magnification of x 500. The substance is purified by
(30931-67-0) treating with hydrochloric acid and washing with water.
Chromogenic substrate suitable for use in EUSA procedures. Particle size Maximum 5% is retained on a sieve No.
Green tablets, freely soluble in water. 180. Maximum 10% passes a sieve No. 125.
pH (2.2.3) 4.2 to 5.8 for a 0.1 gIL solution. Diatomaceous Support, Alkali-washed
Diammonium Hydrogen Orthophosphate Arnmonium Diatomaceous support that has been treated with potassium
phosphate; (NH4)zHP0 4 = 132.1 (7783-28-0) hydroxide solution to reduce peak-tailing of basic
compounds.
2014 Appendix 1 A V -AS 1
Diatomaceous Support, Silanised Diatomaceous earth Dichlofenthion C lO H 13 Cl z0 3PS = 315.2 (97-17-6)

for gas chromarography, silanised A suitable certified reference solution (10 ng/¡.tL in
Acid-washed diatomaceous support silanised with cyclohexane) may be used.
dimethyldichlorosilane or other suitable silanising agents. Dichloroacetic Acid C 2H zCl zOz = 128.9 (79-43-6)
Diazinon Dimpylate; C12H21N203PS = 304.3 (333-41-5) Colourless liquid, miscible with water and ethanol (96%).
Boiling point, about 306°. d~g, about 1.566; ng>, about 1.466; boiling point, about
A suitable certified reference solution (10 ng/¡.tL in iso- 193°.
octane) may be used. Dichloroacetic Acid Solution Dilute 67 mL of
Diazobenzenesulfonic Acid Solution dichloroacetic acid ro 300 mL with water and neutralise ro blue
Diazobenzenesulphonic acid solution litmus paper using ammonia. Cool, add 33 mL of dichloroacetic
Heat 0.2 g of sulfanilic acid with 20 mL of 1M hydrochloric acid and dilute to 600 mL with water.
acid until dissolved, cool to about 4° and add, dropwise, 3,5-Dichloroaniline 3,5-Dichlorophenylamine;
2.2 mL of a 4% w/v solution of sodium nitrite, swirling C 6H ,CI2N = 162.0 (626-43-7)
continuously. AlIow ro stand in ice for 10 minutes and add Melting point, 46° to 52°.
1 mL of a 5% w/v solution of sulfamic acid. Dichlorobenzene See 1,2-Dichlorobenzene.
Diazobenzenesulfonic Acid Solution Rl 1,2-Dichlorobenzene C 6H 4Clz = 147 .0 (95-50-1)
Diazobenzenesulphonic acid solution Rl
Colourless, oily liquid, practically insoluble in water, soluble
Dissolve 0.9 g of sulfanilic acid in a mixture of 30 mL of in anhydrous ethano!.
dilute hydrochloric acid and 70 mL of water. To 3 mL of the
d~g, about 1.31; boiling point, about 180°.
solution add 3 mL of a 5% w/v solution of sodium nitrite.
Cool in an ice-bath for 5 minutes, add 12 mL of the sodium 2,3-Dichloro-5,6-dicyanobenzoquinone 4,5-Dichloro-
nitrite solution and cool again. Dilute to 100 mL with water 3,6-dioxo-cyclohexa-l,4-diene-l,2-dicarbonitrile;
and keep the reagent in an ice-bath. Prepare C S CI2N zO z = 227.0 (84-58-2)
extemporaneously but allow ro stand for 15 minutes before Yellow or orange crystals, soluble in dioxan and in acetic
use . acid, slightly soluble in methylene chloride. lt decomposes in
Dibenzosuberone Dibenzo [a,d] cyclohepta-l ,4-dien-3-one; water.
10, ll-dihydro-5H-dibenzo [a,d] cyclohepten-5-one; Melting point, about 214°.
C 1s H 12 0 = 208.3 (1210-35-1) Srore at a temperature of 2° to 8°.
General reagent grade of commerce. (S) -3,5-Dichloro-2,6-dihydroxy-N-[ (l-ethylpyrrolidin-
Melting point, about 34°. 2-yI)methyI]benzamide hydrobromide
Dibutylamine See Di-n-butylamine. C14H19BrClz03 = 44l.l (113310-88-6)
Di-n-butylamine N- Butylbutan-l-amine, dibutylamine; White or almost white, crystalline powder.
CSH1 9N = 129.3 (111-92-2) [a]jS2, +11.4, determined on a 15.0 gIL solution in anhydrous
Colourless liquid. ethanol.
n5°, about 1.417; boiling point, about 159°. Melting point, about 212°.
Dibutylammonium Phosphate for Ion-pairing 1,2-Dichloroethane Ethylene chloride; C 2 H 4 Cl z = 98.96
(107-06-2)
A colourless solution of 10% ro 15% VN of di-n-butylamine
and 12% to 17% VN of phosphoric acid in water, suitable Clear, colourless liquid, soluble in about 120 parts of water
for ion-pairing in liquid chromatography. and in 2 parts of ethanol (96%).
Dibutyl Ether CSH1SO = 130.2 (142-96-1) d~g, about 1.25.
Colourless, ftammable liquid, practically insoluble in water, Distillation range (2.2.11). Not less than 95 % distils
miscible with anhydrous ethano!. between 82° and 84°.
d~g, about 0.77; ng>, about 1.399. Dichlorofluorescein See 2,7-Dichlorofiuorescein.
Do not distil if the dibutyl ether does not comply with the test jor 2,7-Dichlorofluorescein Dichloroftuorescein, 2-(2,7-
peroxides. dichloro-6-hydroxy-3-oxo-3H-xanthen-9-yl) benzoic acid;
Peroxides. Place 8 mL of potassium iodide and starch CZoHlOClzOs = 401.2 (76-54-0)
SOlUti011 in a 12 mL ground-glass-sroppered cylinder about Yellowish-brown or yellow-orange powder, slight1y soluble in
1.5 cm in diameter. Fill completely with the substance ro be water, freely soluble in ethanol (96%) and in dilute solutions
examined, shake vigorously and allow ro stand protected of alkali hydroxides giving a solution showing a yellowish-
from light for 30 minutes. No colour is produced. green ftuorescence.
The name and concentration of any added stabiliser are 5,7 -Dichloro-8-hydroxyquinoline 5,7 -Dichloroquinolin-
stated on the labe!' 8-01; CgHsClzNO = 214.1 (773-76-2)
Dibutyl Phthalate Di-n-butyl phthalate; General reagent grade of commerce.
C1 6H2204 = 278.3 (84-74-2) Melting point, about 181 0 .
Clear, colourless or faintly coloured, oily liquid, very slight1y

Dichloromethane Methylene chloride; CH 2 CI 2 = 84.9
soluble in water, miscible with acerone and with ethanol
(75-09-2)
(96%).
d~g, 1.043 to 1.048; n5°, 1.490 to 1.495.
Colourless liquid, sparingly soluble in water, miscible with
ethanol (96%).
Dicarboxidine Hydrochloride 4,4'-[(4,4'-
Diaminobiphenyl-3,3'-diyl)dioxy] dibutanoic acid Boiling point, 39° to 42°.
dihydrochloride; C2oHz4Nz06,2HCI = 461 .3 (56455-90-4)
Methylene chloride used in fluorimetry complies with the Melting point, about 179°.
following additional test. Content, minimum 95.0%.
Fluorescence Under irradiation at 365 nm, the Dichloroquinonech1orimide See 2,6-Dichloroquinone-4-
fluorescence (2.2.21) measured at 460 nm in a 1 cm cell is chloroimide.
not more intense than that of a solution containing 2,6-Dich1oroquinone-4-chloroimide
0.002 ppm of quinine in 0.5 M sulfunc acid measured in the Dichloroquinonechlorimide; C 6H 2 CI 3NO = 210 .4
same conditions. (101-3 8-2)
Dich1oromethane, Acidified Acidified methylene Pale yellow or greenish-yellow crystalline powder, practically
ch10ride insoluble in water, soluble in ethanol (96%) and in dilute
To 100 mL of dichloromethane add 10 mL of hydrochlonc acid, alkaline solutions.
shake, allow to stand and separate the two layers. Use the Melting point, about 66°.
lower layer.
Dichlorvos 2,2-Dichlorovinyl dimethyl phosphate;
Dich1oromethane IR C 4H 7Cl 20 4P = 221 (62-73-7)
Spectroscopic reagent grade of commerce. Colourless or brownish-yellow liquid, soluble in water,
Dich1oromethane Reagent Add 50 g of sodium hydrogen miscible with most organic solvents.
carbonate to 1000 mL of dichloromethane, allow to stand nfi, about 1.452.
overnight and filter.
Di-2-cyanoethyl Ether (2-Cyanoethyl) ether;
2,4-Dich1oro-l-naphthol C IO H 6CI20 = 21 3. 1 (2 050- C 6HgN 20 = 124 .1 (1656-48-0)
76-2)
Chromatographic grade of commerce.
Boiling point, about 111 0; n~, 1.4400 .
Dicyc10hexyl Bicyclohexyl; C 12H 22 = 166.3 (92-51-3)
2,6-Dich1orophenol C 6H 4ChO = 163.0 (87-65-0)
d5g, about 0.864; boiling point, about 227°; melting point,
about 4°.
Dich1orophenolindophenol, Sodium Salt Dicyc10hexylamine N,N-Dicyclohexylamine;
See 2,6-Dichlorophenolindophenol sodiwn salto C 12H 23 N = 181.3 (101-83-7)
2,6-Dich1orophenolindophenol Sodium Salt The Colourless liquid, sparingly soluble in water, miscible with
sodium derivative of 2,6-dichloro-N-(4-hydroxyphenyl)-I,4- the usual organic solvents.
benzoquinone monoimine; Tillman's reagent;
n~, about 1.484; boiling point, about 256°; freezing point, 0
0
C1 2H 6C12NNa0 2' 2H 20 = 236. 1 (620-45-1)

to 1°.
Dark-green powder, freely soluble in water and in anhydrous
Dicyc10hexylurea See 1,3-Dicyclohexylurea.
ethano!. The aqueous solution is dark blue; when acidified it
becomes pink. 1,3-Dicyc1ohexylurea C I3 H 24N 20 = 224.4 (2387-23-7)
2,6-Dich1orophenolindophenol Solution Warm 0.1 g of White or almost white, crystalline powder.
2,6-dichlorophenolindophenol sodium salt with 100 mL of water Melting point, about 232°.
and filter. Didocosahexaenoin Diglyceride of docosahexaenoic acid
U se within 3 days of preparation. (C22:6); Glycerol didocosahexaenoate; (all-Z)-
2,6-Dich1orophenolindophenol Solution, Double- Docosahexaenoic acid, diester with propane-l ,2,3- triol;
strength Standard Dissolve 0.1 g of 2,6- C47H6g0 S = 713.0 (88315-12-2)
dichlorophenolindophenol sodium salt in 100 mL of water, filter, The reagent from Nu-Chek Prep, lnc. has been found
standardise by the method described under standard suitable.
dichlorophenolindophenol solution and dilute the solution with DidodecyI3,3 '-thiodipropionate C30Hsg0 4S = 514.8
water so that 1 mL is equivalent to 0.2 mg of ascorbic acid. (123-28-4)
Use within 3 days of preparation and standardise White or almost white, crystalline powder, practically
immediately before use. insoluble in water, freely soluble in acetone and in light
Dich1orophenolindophenol Solution, Standard petroleum, slight1y soluble in ethanol (96%).
Dissolve 50.0 mg of dichlorophenolindophenol, sodium salt in Melting point, about 39°.
100.0 mL of water and filter. Dieldrin C 12H gCl 60 = 380.9 (60-57-1)
Assay Dissolve 20.0 mg of ascorbic acid in lO mL of a Boiling point, about 385°; melting point, about 176°.
freshly prepared 20% w/v solution of metaphosphonc acid and
dilute to 250.0 mL with water. Titrate 5.0 mL rapidly with A suitable certified reference solution (10 ng/~L in
the dich1oro-phenolindophenol standard solution, added cyclohexane) may be used.
from a microburette graduated in 0.01 mL, until the pink Diethanolamine 2,2 '-Iminobisethanol;
colour persists for 10 seconds, the titration occupying not C 4H l1 N0 2 = 105.1 (111-42-2)
more than 2 minutes. Dilute the dichlorophenolindophenol Viscous, clear, slightly yellow liquid or deliquescent crystals
solution with water to make 1 mL of the solution equivalent melting at about 28°, very soluble in water, in acetone and in
to 0.1 mg of ascorbic acid (C 6Hg0 6). methano!.
Use within 3 days. d5g, about 1.09.
Standardise immediately before use. pH (2.2.3) 10.0 to 11.5 for a 5.0% w/v solution.
S, 7-Dich1oroquinolin-8-o1 5,7 -Dichlorooxine; Diethanolamine used in the test for alkaline phosphatase complies
C 9 H sCI 2NO = 214.1 (773-76-2) with the following additional test.
Yellow, crystalline powder, soluble in acetone, slight1y soluble Ethanolamine Not more than 1.0% w/w when
in ethanol (96 %). determined in the following manner. Dissolve 1.00 g of 3-
2014 Appendix 1 A V-AS3
aminopropanol (internal standard) in sufficient acetone 10 dig, 0.827; boiling point, about 146°.
produce 10 mL (solution A). Dissolve 0.5 g of ethanolamine Water (2.5.12) Maximum 1.0%, deterrnined on 0.500 g.
in sufficient acetone 10 produce 10 mL (solution B) . Carry Di(2-ethylhexyl) Phthalate Di(2-ethylhexyl) benzene-1,2-
out the method for gas chromatography, Appendix III B, dicarboxylate, dioctyl phthalate; C24H3S04 = 390.5
using the following solutions. For solution (1) add 1 mL of (117-81 -7)
solution A to 5 g of the substance being examined and dilute
10 10 mL with acetone. For solution (2) dissolve 5 g of the Colourless, oily liquid, practically insoluble in water, soluble
substance being examined in sufficient acetone to produce in organic solvents.
10 mL. For solution (3) add 1 mL of solution A to 0.5 mL dig, about 0.98; ni>°, about 1.486.
of solution B and dilute to 10 mL with ace1One. Prepare Viscosity (2.2.9): about 80 mPa·s.
solution (4) in the same manner as solution (3) but adding Diethylphenylenediamine Sulfate See N,N-Diethyl-p-
1 mL of solution B in place of 0.5 mL of solution B. phenylenediamine sulfate.
Prepare solution (5) in the same manner as solution (3) but
Diethyl Phthalate ClzHl404 = 222.2 (84-66-2)
adding 2 mL of solution B in place of 0.5 mL of solution B.
The chromatographic procedure may be carried out using a
column (1 m x 4 mm) packed with diphenylphenylene Boiling point, about 298°.
oxidepo/ymer (180 to 250 ~lm) and using 40 mL per minute N,N-Diethyl-p-phenylenediamine Sulfate N,N-Diethyl-
as the flow rate of the carrier gas. Maintain the temperature p-phenylenediamine sulphate, diethylphenylenediamine
0
of the column at 125 for 3 minutes and then raise to 300 at 0
sulfate, diethylphenylenediamine sulphate;
arate of 12° per minute. Maintain the temperature of the ClOHI6NZ,HzS04 = 262.3 (6283-63-2)
injection port at 250 0 and that of the detector at 280 0 • White or slightly yellow powder, soluble in water.
Diethoxytetrahydrofuran See 2,5-Diethoxytetrahydrofuran. Melting point, about 185°, with decomposition.
2,5-Diethoxytetrahydrofuran Diethoxytetrahydrofuran, a Store protected from light.
mixture ofthe cis and trans isomers; C SH l6 0 3 = 160.2 Diethylphenylenediamine Sulfate Solution
(3320-90-9) Diethylphenylenediamine sulphate solution
Clear, colourless or slightly yellowish liquid, practically To 250 mL of water add 2 mL of sulfuric acid and 25 mL of
insoluble in water, soluble in ethanol (96%) and in most 0.02M disodium edetate. Dissolve in this solution 1.1 g of
other organic solvents. N,N-diethyl-p-phenylenediamine sulfate and dilute to 1000 mL
dig, about 0.98; n~, about 1.418. with water.
Diethylamine C 4 H ll N = 73.14 (109-89-7) Do not use if the solution is not colourless.
Clear, colourless, flammable liquid, strongly alkaline, S10re protected from light and heat for 1 month.
misciblewith water and with ethanol (96%). Diflubenzuron 1-(4-Chlorophenyl)-3-(2,6-
dig, about 0.71; boiling point, about 55°. difluorobenzoyl)urea; Cl4H9CIFzNzOz = 310.7 (35367-38-5)
Diethylamine Rl C 4H l1 N = 73.1 (109-89-7) Colourless or white or almost white crystals, practically
Content, minimum 99.5%. insoluble in water, freely soluble in dimethyl sulfoxide,
Clear, colourless, flammable liquid, strongly alkaline, miscible slightly soluble in acetone.
with water and with ethanol (96%). Melting point, 230° to 232°.
dig, about 0.71; boiling point, about 55°. Digitonin CS6H920Z9 = 1229 (11024-24-1)
Diethylaminoethyldextran Anion exchange res in Crystals, practically insoluble in water, sparingly soluble in
presented as the hydrochloride. anhydrous ethanol, slightly soluble in ethanol (96%) .
Powder forming gels with water. Digitoxin C41H64013 = 765 .0 (71-63-6)
N,N-Diethylaniline C1oH1SN = 149.2 (91-66-7) Of the British Pharrnacopoeia.
dig, about 0.938; boiling point, about 21T; melting point, Digoxin Reagent Add 98 mL of glacial acetic acid 10 2 mL
about - 38°. of suljuric acid and add 0.1 mL of a 5% w/v solution of
Diethylene Glycol Digol; 2,2'-oxydiethanol; anhydrous iron(III) chloride in glacial aceu'c acid.
C4H 10 0 3 = 106 .1 (111-46-6) Dihydrocapsaicin N- [(4-Hydroxy-3-
Content, minimum 99.5% mlm. methoxyphenyl)methyl]-8-methylnonanamide;
CIsHz9N03 = 307.4 (19408-84-5)
Clear, colourless liquid, hygroscopic, miscible with water,
with acetone and with ethanol (96%). White or almost white, crystalline powder, practically
insoluble in cold water, freely soluble in ethano!.
dig, about 1.118; ni>°, about 1.447; boiling point, 244° 10
246°. lO,ll-Dihydrocarbamazepine 10,11-Dihydro-5H-
dibenzo[bJ]azepine-5- carboxarnide; C1sHl4NzO = 238 .3
Store in an airtight container. (3564-73-6)
N,N-Diethylethane-l,2-diamine See Melting point, 205° to 210°.
N,N-Diethylenediamine.
Dihydrocarvone p-Menth-8-en-2-one; 2-Methyl-5-
N,N-Diethylethylenediamine N,N-Diethyl-1,2- (l-methylethenyl)cyc1ohexanone; C lOH l6 0 = 152.2
diaminoethane; C 6H l6 N 2 = 116.2 (100-36-7) (7764-50-3)
Content, minimum 98.0% . Dihydrocarvone used in gas chromatography complies with the
Slightly oily liquid, colourless or slightly yellow, strong odour following additional test.
of arnmonia, irritant to the skin, eyes and mucous Assay Gas chromatography (2.2.28) as prescribed in the
membranes. test for chromatographic profile in the monograph Caraway
oil (1817) .
Content, ca1culated by the nonnalisation procedure: major The name and concentration of any added stabiliser are
component (trans-dihydrocarvone): minimum 70 %; sum of stated on the labe!'
cis- and trans-dihydrocarvone: minimum 98%. Srore protected from light.
1,8-Dihydroxyanthraquinone Danthron; dantron, Di-isopropyl Ether, Stabiliser-free Di-isopropyl ether that
dihydroxyanthraquinone; C¡ 4HS04 == 240.2 (117-10-2) is free from stabiliser.
Crystalline orange powder, practically insoluble in water, . Reagent grade of commerce.
slightly soluble in ethanol (96%), soluble in solutions of alkali Di-isopropylethylamine Ethyldi-isopropylamine;
hydroxides. C SH 20N 2 = 144.3 (7087-68-5)
Dantron used in the sesquiterpenic acids assay in Valerian root Boiling point, about 121"; nbo, abour 1.41 4.
(0453) complies with the following additional tests.
N,N' - Di-isopropylethylenediamine N,N'-
A(1 %, 1 cm), 355 ro 375, detennined at 500 nm in 1 M bis(I-Merhylethyl)-1,2-ethanediamine; C S H 20 N 2 = 144.3
potassium hydroxide. (4013-94-9)
Assay Liquid chromatography (2.2.29) as prescribed in the Colourless or yellowish, corrosive, ftammable, hygroscopic
monograph Valerian R oot (0453) at the concentration of the liquido
reference solution.
Content, minimum 95%, ca1culated by the normalisation
d~g, about 0.798; ni:?,
abour 1.429; boiling point, about
170°.
procedure.
Dillapiole 6-AlIyl-4,5-dimethoxy-l,3-benzodioxole;
2,5-Dihydroxybenzoic Acid Gentisic acid; C¡ 2H¡ 40 4 == 222.24 (523-80-8)
C 7 H 6 0 4 == 154.1 (490-79-9)
General reagenr grade of commerce.
Light yellow crystals. Melting point, about 200°.
2,5-Dimethoxybenzaldehyde C SH IO 0 3 == 166 .2 (93-02-
5,7 -Dihydroxy-4-methylcoumarin 5,7 -Dihydroxy-4- 7)
methyl-2H-l-benzopyran-2-one; C 1oH g0 4 = 192.2
(2 107-76-8)
Light yellowish powder, practically insoluble in water, Melring poinr, about 50 c •
sparingly soluble in ethanol (96%). 4,4 ' -Dimethoxybenzophenone
Melting point, 295° ro 303°. bis(4-Merhoxyphenyl)methanone; C1sH ¡40 3 == 242.3 (90-
96-0)
Dihydroxynaphthalene See Naphthalene-1,3-diol.
White or almost white powder, practically insoluble in water
1,3-Dihydroxynaphthalene See N aphthalene-1, 3-diol. and slightly soluble in ethanol (96 %).
2,7-Dihydroxynaphthalene See Naphthalene-2,7-diol. Melting point, about 142 c •
2,7-Dihydroxynaphthalene Solution S ee Naphthalenediol 3,4-Dimethoxyphenethylamine C IO H 1SN0 2 == 181.2
solution.
(120-20-7)
5,7-Di-iodo-8-hydroxyquinoline 5,7 -Di-iodoquinolin-8- General reagent grade of commerce.
01; C 9 H s I 2NO == 397.0 (83- 73-8)
An oily liquid; nbo, about 1.546; weight per mL, abour
Yellowish-brown powder, sparingly soluble in acetone and in 1.07 g.
ethanol (96%).
Dimethoxypropane See 2,2-Dimethoxy propane.
2,2-Dimethoxypropane Dimethoxypropane;
1,5-Di-iodopentane C sH 1oI2 == 323.9 (628-77-3) C SH 12 0 2 = 104. 1 (77-76-9)
General reagent grade of commerce. Colourless liquid, decomposing on exposure ro moist air or
A colourless liquid; boiling point, abo ut 101 °. water.
5,7-Di-iodoquinolin-8-o1 See 5,7-Di-iodo-8- d~g, about 0.847; nbo, about 1.378; boiling point, about 83°.
hydroxyquinoline
Dimethylacetamide C 4 H 9 NO == 87.1 (127-19-5)
Di-isobutyl Ketone 2,6-Dimethyl-4-heptanone; Content, minimu.m 99.5%.
C 9 H 1S O == 142.2 (108-83-8)
Colourless liquid, miscible with water and with many organic
Clear, colourless liquid, slightly soluble in water, miscible solvents.
with most organic solvents.
nbo, about 1.414; boiling point, about 168 e .
d~g, about 0.94; ni:?,
about 1.437; boiling point, about 165°.
Dimethylamine N-methylmethanamine; C 2H 7 N = 45.08
Di-isopropyl Ether Isopropyl ether; C 6 H I4 0 == 102.2 (124-40-3)
(108-20-3)
Colourless, ftammable gas; boiling point, about 7"; melting
Clear, colourless liquid, very slightly soluble in water, point, about - 92.2°.
miscible with ethanol (96 %) .
Dimethylamine Hydrochloride C 2 H s ClN == 81.6
d~g, 0.723 ro 0.728; boiling point, 67° ro 69°.
(506-59-2)
Do not distil ¡I the di-isopropyl ether does not comply with the test
for peroxides.
Melting point, about 172 c .
Peroxides Place 8 mL of potassium iodide and starch
solution in a 12-mL ground-glass-stoppered cylinder about
Dirnethylamine solution
1.5 cm in diameter. Fill completely with the substance to be A 400 gIL solution.
examined, shake vigorously and allow ro stand protected Clear, colourless solution.
from light for 30 minutes. No colour is produced. Density, about 0.89; boiling point, about 54' ; melting point,
00
about - 37 •
Dimethylaminobenzaldehyde See Yellow, crystalline powder, slightly soluble in water, soluble

4-Dimethylaminobenzaldehyde. in methanol.
4-Dimethylaminobenzaldehyde Melting point, about 70°.
Dimethylaminobenzaldehyde; C 9 H l1 NO = 149.2 (100-10-7) 3-DimethylaminophenoI 3-(Dimethylamino)phenol;
White or yellowish-white crystals, soluble in ethanol (96%) CsHIINO = 137.2 (99-07-0)
and in dilute acids. Grey powder, slightly soluble in water.
Melting point, about 74°. Melting point, about 80°.
Dimethylaminobenzaldehyde Reagent Dissolve 0.5 g of 2- (Dimethylamino )thioacetamide hydrochloride
4-dimethylaminobenzaldehyde in a cooled mixture of 53 mL of C 4 H l1 CIN 2S = 154.7 (27366-72-9)
sulfurie acid and 50 mL of water and add 0.5 mL of iron(m) Dimethylaniline See N,N-Dimethylaniline.
ehloride soluríon R 1.
N,N- Dimethylaniline Dimethylaniline; CsHIIN = 121.2
Allow to stand for 2 hours before use. (121-69-7)
Dimethylaminobenzaldehyde Solution, Alcoholic Clear, oily liquid, almost colourless when freshly distilled,
Dissolve 1 g of 4-dimethylaminobenzaldehyde in 30 mL of darkening on storage to reddish-brown, practically insoluble
ethanol (96%) and add 180 mL of butan-1-ol and 30 mL of in water, freely soluble in ethanol (96%).
hydroehlorie acid.
n~, about 1.568.
Prepare immediately before use and discard if a pink colour
develops. Distillation range (2.2.11) Not less than 95% distils
between 192° and 194°.
Dimethylaminobenzaldehyde Solution Rl Dissolve
0.2 g of dimethylaminobenzaldehyde in 20 mL of ethanol 2,3-Dimethylaniline 2,3-Xylidine; C SH l1 N = 121.2 (87-
59-2)
(96%) and add 0.5 mL of hydroehlorie acid. Shake the
solution with activated eharcoal and filter. The colour of the Yellowish liquid, sparingly soluble in water, soluble in
reagent is less intense than that of iodine solution R3. Prepare ethanol (96%).
irnmediately before use. nt
dig, 0.993 to 0.995; O, about 1.569; boiling point, about
Dimethylaminobenzaldehyde Solution ID Dissolve 224°.
0.2 g of dimethylaminobenzaldehyde, without heating, in a 2,4-Dimethylaniline 2,4-Xylidene; CSH11N = 121.2
mixture of 4.5 mL of water and 5.5 mL of hydroehlorie acid. (95-68-1)
Prepare immediately before use. General reagent grade of commerce.
Dimethylaminobenzaldehyde Solution R6 Dissolve 2,6-Dimethylaniline 2,6-Xylidine; CsH1IN = 121.2
0.125 g of dimethylaminobenzaldehyde in a cooled mixture of (87-62-7)
35 mL of water and 65 mL of sulfurie acid. Add 0.1 mL of a Colourless liquid, sparingly soluble in water, soluble in
5% w/v solution of Jerrie ehloride. Before use allow to stand ethanol (96%) .
for 24 hours, protected from light.
When stored at room temperature, use within 1 week; when
dig, about 0.98.
stored in a refrigerator, use within several months. 2,6-Dimethylaniline Hydrochloride 2,6-
Dimethylbenzenamide hydrochloride; 2,6-Xylidine
Dimethylaminobenzaldehyde Solution R7 Dissolve hydrochloride; CsHl zCIN = 157.6 (21436-98-6)
1.0 g of dimethylaminobenzaldehyde in 50 mL of hydroehlorie
aeid and add 50 mL of ethanol (96%). Content, minimum 98.0%.
Store protected from light; use within 4 weeks. 2,4-Dimethyl-6-tert-butylphenol 6-tert-Butyl-2,4-
dimethylphenol; C 12 H 1S O = 178.3 (1879-09-0)
Dimethylaminobenzaldehyde Solution R8 Dissolve
0.25 g of dimethylaminobenzaldehyde in a mixture of 5 g of General reagent grade of commerce.
phosphorie acid, 45 g of water and 50 g of anhydrous aeetic Dimethyl Carbonate C 3H 60 3 = 90.1 (616-38-6)
acid. Prepare immediately before use. Liquid, insoluble in water, miscible with ethanol (96%).
4-Dimethylaminocinnamaldehyde C II H 13NO = 175.2 dl 7 , about 1.065; n~, about 1.368; boiling point, about 90°.
(6203-18-5) Dimethyl-~-cyclodextrin Heptakis(2,6-di-O-
Orange or orange-brown crystals ar powder. Sensitive to methyl)cyc1omaltoheptaose; Cyc1oheptakis-( 1-+ 4)-(2,6-di-O-
light. methyl-ex-D-glucopyranosyl) ;
Melting point, about 138°. 2A,2 B,2c,2D,2E,2F,2G,6A,6B,6c ,6 D,6 E,6 F,6G-Tetradeca-O-
methyl- ~- cyc1odextrin; C56H98035 = 1331 (51166-71-3)
4-Dimethylaminocinnamaldehyde Solution Dissolve
2 g of 4-dimethylaminocinnamaldehyde in a mixture of 100 mL White or almost white powder.
of hydroehlorie aeid R1 and 100 mL of anhydrous ethanol. Dimethyldecylamine C I2 H 27 N = 185.4 (1120-24-7)
Dilute the solution to four times its volume with anhydrous Content, minimum 98.0% w/w.
ethanol irnmediately befare use.
Boiling point, about 234°.
2- (Dimethylamino) ethyl Methacrylate N,N- Dimethyldodecylamine N-oxide
2-(Dimethylamino) ethyl 2-methylpropenoate; C l4 H 31NO = 229.4 (1643-20-5)
C SH 1SN0 2 = 157.2 (2867-47-2)
d¡O, about 0.930; boiling point, about 187°. Melting point, about 132°.
Dimethylaminonaphthalenesulfonyl Chloride
Dimethylaminonaphthalenesulphonyl chloride; 5- l,l-Dimethylethylamine 2-Amino-2-methylpropane; terc-
dimethylaminonaphthalene-1-sulfonyl chloride; 5- butylamine; C 4 H 11 N = 73.1 (75-64-9)
dimethylaminonaphthalene-1-sulphonyl chloride; dansyl Liquid, miscible with ethanol (96%).
chloride; CI 2HI 2CIN02S = 269.8 (605-65-2) nt
dig, about 0.694; O, about 1.378; boiling point, about 46°.
V-AS6 Appendix 1 A 2014
l,l-Dimethylethyl Methyl Ether tert-Butyl rnethyl ether; Dimethyl Phthalate C IO H IO 0 4 = 194.2 (131-11-3)
C sH 12 0 = 88.1 (1634-04-4) General reagem grade of commerce.
Colourless, c1ear, fiarnrnable liquido A colourless or faintIy coloured liquid; weight per mL, about
nbo, about 1.376. 1.19 g.
Minimum transmittance (2.2.25) Using water as Dimethylpiperazine See N,N' -Dimethylpiperazine.
cornpensation liquid: 50% at 240 nrn, 80% at 255 nrn, 98% N,N'-Dimethylpiperazine 1,4-Dimethylpiperazine;
at 280 nm. C óH I4N z = 114.2 (106-58-1)
l,l-Dimethylethyl Methyl Ether Rl A colourless liquid, miscible with water and with ethanol
Content, minimum 99.5%. (96%).
d~g, about 0.741; n~, about 1.369; boiling point, about 55°. d~g, about 0.85; n~, about 1.446; boiling poim, about 131 °.
Dimethylformamide C 3H 7NO = 73.1 (68-12-2) Dimethylstearamide See Dimethylstearylamide.
Clear, colourless neutralliquid, miscible with water and with Dimethylstearylamide Dirnethylsteararnide; N,N-
ethanol (96%). dimethyloctadecanamide; C ZO H 41NO = 311.5
d~g, 0.949 to 0.952; boliling point, about 153°. White or almost white solid mass, soluble in many organic
Water (2.5.12) Maximum 0.1%. solvents, inc1uding acetone.
Dimethylformamide Diethylacetal C7H17NOz = 147.2 Melting poim, about 51 0 .
(1188-33-6) Dimethyl Sulfone Dimethyl sulphone; CzHóOzS = 94.13
nbo, about 1.40; boiling poim, 128° to 130°. (67-71-0)
N,N-Dimethylformamide Dimethylacetal 1,1- White or almost white, crystaIline powder, freely soluble in
Dimethoxytrimethylamine; CsH13NOz = 119 .2 (4637-24-5) water, soluble in acetone and ethanol (96%) .
Clear, colourless liquid. Melting point, 108° to 110°.
d~g, about 0.896; nbo, about 1.396; boiling poim, about Dimethyl Sulfoxide Dimethyl sulphoxide;
103°. CzHóOS = 78.1 (67-68-5)
Dimethylglyoxime 2,3-Butanedione dioxime; Of the British Pharmacopoeia.
C 4H sN zO z = 116.1 (95-45-4) Dimethyl sulfoxide used in spectrophotometry complies with the
White or almost white, crystaIline powder or colourless following additional test.
crystals, practicaIly insoluble in cold water, very slightIy Minimum transmittance (2.2.25) Using water as
soluble in boiling water, soluble in ethanol (96%) . compensation liquid : 10% at 262 nm, 35% at 270 nm, 70%
MeIting point, about 240°, with decomposition; sulfated ash, at 290 nm, 98% at 340 nm and at higher wavelengths.
not more than 0.05%. Dimethyl Sulfoxide Rl Dimethyl sulphoxide Rl Comains
1,3-Dimethyl-2-imidazolidinone CsHIONzO = 114.2 not less than 99 .7% of CzHóOS, determined by gas
(80-73-9) chromatography.
nbo, about 1.4720; boiling poim, about 224°. N,N-Dimet;hyItetradecylamine C 1óH 3S N = 241.5
N,N-Dimethyl-p-nitrosoaniline CsHIONzO = 150.2 General reagem grade of commerce comaining 98.0 to
(138-89-6) 101.0% w/w of C 1óH 3SN.
General reagem grade of commerce. A c1ear or almost c1ear, colourless or slightIy yeIlow liquid;
Green crystals or a green, crystaIline powder; melting poim, boiling point, about 260°; d~g, about 0.80.
about 86°. Complies with the following tests.
N,N-Dimethyloctylamine Octyldimethylamine; Water Not more than 0.3%, Appendix IX C, Method I A.
C 10H Z3N = 157.3 (7378-99-6) Assay Dissolve 0.2 g in 10 mL of ethanol (96%) and titrate
Colourless liquido with O.IM hydrochloric acid VS using methyl red solution as
d~g, about 0.765; n~, about 1.424; boiling poim, about
indicator until a red colour is produced. Each mL of
195°. O.IM hydrochloric acid VS is equivalent to 24.15 mg of
C 1óH 3SN.
2,5-Dimethylphenol p-Xylenol; CsHIOO = 122.2 (95-87-4)
Dimethyl YelIow CI 11020; 4-dimethylaminoazobenzene;
White or almost white crystals.
CI4H1SN3 = 225.3 (60-11-7)
2,6-Dimethylphenol CsHIOO = 122.2 (576-26-1)
Produces a red colour in moderately acidic aJcoholic
Colourless needles, slightIy soluble in water, very soluble in
solutions and a yeIlow colour in weakIy acidic and alkaline
ethanol (96%).
solutions (pH range, 2.8 to 4.6).
Boiling poim, about 203°; melting poim, 46° to 48°.
3,4-Dimethylphenol CsH100 = 122.2 (95-65-8)
White or almost white crystals, slightIy soluble in water, Homogeneity Carry out the method for thin-layer
freely soluble in ethanol (96%) . chromatography, Appendix III A, using silica gel G as the
coating substance and dichloromethane as the mobile phase.
Boiling poim, about 226°; melting point, 25° to 27°.
Apply to the plate 10 ¡.tL of a 0.01% w/v solution in
N,N-Dimethyl-L-phenylalanine (2S)-2-(Dimethylamino)- dichloromethane. The chrornatogram shows only one spot.
3-phenylpropanoic acid; CllH1SNO z = 193.2 (17469-89-5)
Dimethyl YelIow and Oracet Blue Soludon Dissolve
Melting point, about 226°. 10 rng of dimethyl yellow and 10 rng of oracet blue B in
N,N-Dimethyl-p-phenylenediamine Dihydrochloride 300 rnL of dichloromethane.
C sH 1z N z,2HCI = 209.1 (536-46-9)
General reagent grade of comrnerce.
Darkens readily on exposure to airo
2014 Appendix 1 A V-AS7
Dinlethyl Yellow and Oracet Blue 2R Solution Dissolve Dinitrophenylhydrazine-aceto-hydrochloric Solution

10 mg of dimethyl yellow and 10 mg of oracet blue 2R in Dissolve 0.2 g of dinitrophenylhydrazine in 20 mL of methanol
300 mL of dichloromethane. and add 80 mL of a mixture of equal volumes of acetic acid
Dinlethyl Yellow Solution A 0.2% w/v solution of and hydrochloric acid R1. Prepare imrnediately before use.
dimethyl yellow in ethanol (90%). Dinitrophenylhydrazine-hydrochloric SoIution
Complies with the following test. Dissolve by heating 0.5 g of dinitrophenylhydrazine in dilute
Sensitivity A solution containing 2 g of ammonium chloride hydrochloric acid and complete to 100 mL with the same
solvent. Allow to cool and filter. Prepare immediately before
in 25 mL of carbon dioxide-free water to which is added
0.1 mL of the dimethyl yellow solution is yellow. Not more use.
than 0.10 mL of O.lM hydrochloric acid VS is required to Dinitrophenylhydrazine-sulfuric Acid Solution
change the colour of the solution to red (pH range, 2.8 to Dinitrophenylhydrazine-sulphuric acid solution
4.6) . Dissolve 1.5 g of dinitrophenylhydrazine in 50 mL of a
Dinleticone Of the British Pharrnacopoeia. 20% v/v solution of sulfuric acid. Prepare immediately before
use.
Dinúdium Bromide 3,8-Diamino-5-methyl-6-phenyl-
phenanthridinium bromide; C2oH1 SBrN3 = 380.3 (518-67-2) DinonyI Phthalate C2óH2404 = 418 .6 (28553-12-0)
Dark-red crystals, slightly soluble in water at 20 c , sparingly Colourless to pale yellow, viscous liquid.
soluble in water at 60" and in ethanol (96%). dig, 0.97 to 0.98; n~o, about 1.482 to 1.489.
Dinúdium Bromide-Sulfan Blue Mixed Solution Acidity Shake 5.0 g with 25 mL of water for 1 minute.
Dimidiurn bromide-sulphan blue mixed solution Allow to stand, filter the separated aqueous layer and add
Dissolve separately 0.5 g of dimidium bromide and 0.25 g of 0.1 mL of phenolphthalein solution. Not more than 0.3 mL of
sulfan blue in 30 mL of a hot mixture of 1 volume of O.lM sodium hydroxide VS is required to change the colour of
anhydrous ethanol and 9 volurnes of water, stir, mix the two the solution (0.05%, calculated as phthalic acid).
solutions, and dilute to 250 mL with the same mixture of Water Not more than 0.1 %, Appendix IX C.
solvents. Mix 20 mL of this solution with 20 mL of a 14.0% DioctadecyI Disulfide Dioctadecyl disulphide;
VIV solution of sulftm·c acid previously diluted with about C3óH74S2 ~ 571.1 (2500-88-1)
250 mL of water and dilute to 500 mL with water. White or almost white powder, practically insoluble in water.
Store protected from light. Melting point, 53° to 58°.
Dinitrobenzene See l,3-Dinitrobenzene. 2,2 '-Di(octadecyloxy) -S,5 '-spirobi(1,3,2-
1,3-Dinitrobenzene Dinitrobenzene; C óH 4N 2 0 4 = 168.1 dioxaphosphorinane) C41Hs20óP2 = 733
(99-65-0) White or almost white, waxy sol id, practically insoluble in
Yellowish crystalline powder or crystals, practically insoluble water, soluble in hydrocarbons.
in water, slightly soluble in ethanol (96%). Melting point, 40° to 70°.
Melting point, about 90°. DioctadecyI 3,3 '-Thiodipropionate C42Hs204S = 683
Dinitrobenzene Solution A 1% w/v solution in ethanol (693-36-7)
(96%). White or almost white, crystalline powder, practically
Dinitrobenzoic Acid See 3,5-Dinitrobenzoic acid. insoluble in water, freely soluble in methylene chloride,
3,5-Dinitrobenzoic Acid Dinitrobenzoic acid; sparingly soluble in acetone, in ethanol (96%) and in light
C7H 4 N 20 ó = 212.1 (99-34-3) petroleum.
Almost colourless crystals, slightly soluble in water, very Melting point, 58° to 67°.
solublein ethanol (96%). DioctyI Sodium Sulfosuccinate Dioctyl sodium
Melting point, about 206°. sulphosuccinate, docusate sodium; C2oH37Na07S = 444.6
Dinitrobenzoic Acid Solution A 2% w/v solution in (577-11-7)
ethanol (96%). Docusate sodium of the British Pharrnacopoeia.
Dinitrobenzoyl Chloride 3,5-Dinitrobenzoyl chloride; Dioxan See l,4-Dioxan.
C 7H 3CIN 20 S = 230.6 (99-33-2) 1,4-Dioxan Dioxan; C 4 H g 0 2 = 88.11 (123-91-1)
Translucent, yellow or greenish-yellow powder or yellowish Clear, colourless liquid, miscible with water and with most
crystals, soluble in acetone and in toluene. organic solvents.
Melting point, about 68°. diS, about 1.03; freezing point, 9° to 11 0 .
Suitability test To 1 mL of anhydrous ethanol and 0.1 g of Water (2.5.12), maximum 0.5%.
dinitrobenzoyl chloride add 0.05 mL of dilute sulfuric acid and
Do not distil if the dioxan does not comply with the test for
boil under a reflux condenser for 30 mino After evaporation
on a water-bath add 5 mL of heptane to the residue and heat peroxides.
to boiling. Filter the hot solution. Wash the crystals formed Peroxides Place 8 mL of potassiw11 iod¡de and starch
on cooling to room temperature with a small quantity of solution in a 12 mL ground-glass-stoppered cylinder about
heptane and dry in a desiccator. The crystals melt at 94° to 1.5 cm in diameter. Fill completely with the substance ro be
95°. examined, shake vigorously and allow to stand in the dark
Dinitrophenylhydrazine See 2,4-Dinitropheny lhydrazine. for 30 minutes. No colour is produced.
2,4-Dinitrophenylhydrazine Dinitrophenylhydrazine; Dioxan used for liquid scintillation is of a suitable analytical
C óH óN 40 4 = 198.1 (119-26-6) grade.
Reddish-orange crystals, very slightly soluble in water, slightly Dioxan SoIution Dilute 50.0 mL of dioxan stock solution to
soluble in ethanol (96%). 100.0 mL with water (0 .5 mg per mL of dioxan).
Melting point, about 203°, Appendix V A, Method V.
Dioxan Solution Rl Dilute 10.0 mL of dioxan solution to Orange-yellow, crystalline powder, practically insoluble in
50.0 mL with water (0.1 mg per mL of dioxan). water, freely soluble in ethanol (96%) .
Dioxan Stock Solution Dissolve 1.00 g of dioxan in water Melting point, about 157°, with decomposition.
and dilute to 100.0 mL with the same solvento Dilute Diphenylcarbazone Mercuric Reagent
5.0 mL of this solution to 50.0 mL with water (1.0 mg per Solution A. Dissolve 0.1 g of diphenylcarbazone in anhydrous
mL) .
ethanol and dilute to 50 mL with the same solvent.
Diphenylamine C 12H ll N = 169.2 (122-39-4) Solution B. Dissolve 1 g of mercUl7c chloride in anhydrous
White or almost white crystals, slightly soluble in water, ethanol and dilute to 50 mL with the same solvento
soluble in ethanol (96%). Mix equal volumes of the two solutions.
Melting point, about 55°. 2,2-Diphenylglycine Amino(diphenyl)acetic acid;
Store protected from light. C 14H 13 N0 2 = 227.26 (3060-50-2)
Diphenylamine Solution A 0.1 % w/v solution in sulfuric 1,2-Diphenylhydrazine Hydrazobenzene; 1,2-
acid. Diphenyldiazane; C 12H 12N 2 = 184.3 (122-66-7)
Store protected from light. Orange powder; melting point, about 125°.
Diphenylamine Solution Rl A 1% w/v solution in DiphenyImethanol Benzhydrol; C 13 H 120 = 184.2
sulfuric acid. The solution is colourless. (91-01-0)
Diphenylamine Solution R2 Dissolve 1 g of White or almost white, crystalline powder. Melting point,
diphenylamine in 100 mL of glacial acetic acid and add about 66°.
2.75 mL of sulfuric acid. Use immediately. Diphenyloxazole 2,5-Diphenyloxazole;
Diphenylanthracene See 9,1O-Diphenylanthracene. C1sHllNO = 221.3 (92-71-7)
9,10-Diphenylanthracene Diphenylanthracene; White or almost white powder, practically insoluble in water,
C 26 H 1S = 330.4 (1499-10-1) soluble in methanol, sparingly soluble in dioxan and in glacial
Yellowish or yellow, crystalline powder, practically insoluble acetic acid.
in water. Melting point, about 70°; A(l %, 1 cm), about 1260,
Melting point, about 248°. determined at 305 nm in methanol.
Diphenylbenzidine See N,N'-Diphenylbenzidine. Diphenyloxazole used for liquid scintlilation is of a suitable
N,N'-Diphenylbenzidine Diphenylbenzidine, N,N'- analytical grade.
Diphenylbiphenyl-4,4'-diamine; C24H20N2 = 336.4 Diphenylphenylene Oxide Polymer 2,6-Diphenyl-p-
(531-91-9) phenylene oxide polymer
White or faintly grey, crystalline powder, practically insoluble White or almost white, porous beads. The size range of the
in water, slightly soluble in acetone and in ethanol (96%). beads is specified after the name of the reagent in the tests
Melting point, about 248°. where it is used.
Nitrates Dissolve 8 mg in a cooled mixture of 5 mL of Diphosphorus Pentoxide See Phosphorus pemoxide.
water and 45 mL of nitrogen-free sulfuric acid. The solution is Dipotassium Edetate Dipotassium dihydrogen
colourless or very pale blue. ethylenediaminetetra-acetate; CJOH14N2K20S,2H20 = 404.5
Sulfated ash Not more than 0.1%, Appendix IX A. (25102-12-9)
Store protected from light. General reagent grade of commerce.
Diphenylborlc Acid Aminoethyl Ester Dipotassium Hydrogen Orthophosphate ;Dipotassium
C 14H I6 BNO = 225.1 (524-95-8) hydrogen phosphate; K 2HP0 4 = 174.2 (7758-11-4)
White or slightly yellow, crystalline powder, practically White or almost white, crystalline powder, hygroscopic, very
insoluble in water, soluble in ethanol (96%). soluble in water, slightly soluble in ethanol (96%).
Melting point, about 193°. Store in an airtight container.
Diphenylcarbazide See 1,5-Diphenylcarbazide. Dipotassium Hydrogen Phosphate See Dipotassium
1,5-Diphenylcarbazide Diphenylcarbazide; 1,5- hydrogen orthophosphate.
dipheny1carbonodihydrazide; C 13H 14N 40 = 242.3 (140-22- Dipotassium Hydrogen Phosphate Trihydrate
7) K 2HP0 4,3H 20 = 228.2 (16788-57-1)
White or almost white, crystalline powder which gradually Colourless or white or almost white powder or crystals, freely
becomes pink on exposure to air, very slightly soluble in soluble in water.
water, soluble in acetone, in ethanol (96%) and in glacial Dipotassium Sulfate See Potassium sulfate.
acetic acid. Dipotassium (+)- Tartrate Potassium tartrate;
Melting point, about 170°. C4H4K206,'/2H20 = 235.3 (921-53-9)
Sulfated ash Not more than 0.1 %, Appendix IX A. White or almost white, granular powder or crystals, very
Store protected from light. soluble in water, very slightly soluble in ethanol (96%).
Diphenylcarbazide Solution Dissolve 0.2 g of 2,2'-Dipyridyl 2,2'-Bipyridine; C JOH 8 N 2 = 156.2 (366-
diphenylcarbazide in 10 mL of glacial acetic acid and dilute to 18-7)
100 mL with anhydrous ethanol. Prepare immediately before General reagent grade of commerce.
use. Melting point, about 72°.
Diphenylcarbazone See 1,5-Diphenylcarbazone. 2,2 '-Dipyridylamine N-(pyridin-2-yl)pyridin-2-amine;
1,5-Diphenylcarbazone Diphenylcarbazone; CJOH 9 N 3 = 171.2 (1202-34-2)
C 13H 12N 40 = 240.3 (538-62-5) Melting point, about 95°.
Disodium Arsenate Disodium hydrogen arsenate 5,5' -Dithiobis (2-nitrobenzoic) Acid 3-Carboxy-4-
heptahydrate, dibasic sodium arsenate, sodium arsenate nitrophenyldisulfide, 3-carboxy-4-nitrophenyldisulphide,
heptahydrate; Na2HAs04,7H20 = 312.0 (10048-95-0) D1NB, EIIman's reagent; C¡4HgN20 gS = 396.4 (69-78-3)
Crystals, efflorescent in warm air, freeIy soluble in water, YeIlow powder sparingly soluble in ethanol (96%).
soluble in glycerol, slightIy soluble in ethanol (96%). The Melting point, about 242 0
•
aqueous solution is alcaline to litmus. Dithiol Toluene-3,4-dithiol; 4-methylbenzene-l,2-dithiol;

dig, about 1.87. C 7HgS2 = 156.3 (496-74-2)
Melting point, about 5T, when rapidly heated. White or almost white crystals, hygroscopic, soluble in
Disodium Bicinchoninate 2,2 '-Biquinoline-4-4 '- methanol and in solutions of alkali hydroxides.
dicarboxylate disodium salt, disodium 2,2'-biquinoline-4-4'- Melting point, about 30°.
dicarboxylate; C2oH ION2Na20 4 = 388.3 (979-88-4) Store in an airtight container.
General reagent grade of commerce. Dithiol Reagent Toluenedithiol reagent
Disodium Catechol-3,5-disulfonate 4,5-Dihydroxy-l,3- To 1 g of dithiol add 2 mL of thioglyeollie acid and dilute to
benzene disulfonic acid disodium salt monohydrate, 250 mL with a 2% w/v solution of sodium hydroxide. Prepare
4,5-dihydroxy- I ,3-benzene disulphonic acid disodium salt imrnediately before use.
monohydrate, disodium catechol-3,5- disulphonate;
Dithiothreitol threo-1,4-Dimercapto-2,3-butanediol;
C óH 40 gNa,H 20 = 332 (149-45-1)
C 4H IO 0 2S 2 = 154.2 (27565-41-9)
SlightIy hygroscopic needles, freely soluble in water, in
Disodium Edetate Disodium dihydrogen acetone and in anhydrous ethano!.
ethylenediaminetetra-acetate, dihydrate; sodium edetate;
CIOH¡,¡N2Na20g, 2H20 = 372.2 (6381-92-6)
Dithizone 1,5-Diphenylthiocarbazone;
C13H¡ 2N4S = 256.3 (60-10-6)
Disodium Ethanedisulfonate Disodium
ethanedisulphonate; 1,2-ethanedisulfonic acid disodium salt; A bluish-black, brownish-black or black powder, practicaIly
insoluble in water, soluble in ethanol (96%) .
1,2-ethanedisulphonic acid disodium salt;
C2H4Na20óS2 = 234.2 (5325-43-9) Store protected from light.
General reagent grade of commerce. Dithizone Rl
Disodium Hydrogen Citrate Disodium hydrogen Content, minimum 98.0%.
2-hydroxypropane-1,2,3-tricarboxylate sesquihydrate, sodium BIuish-black, brownish-black or black powder, practicaIly
acid citrate; CóH 6Na207,lV2H20 = 263.1 (144-33-2) insoluble in water, soluble in ethanol (96%).
White or almost white powder, soluble in less than 2 parts of Store protected from light.
water, practicaIly insoluble in ethanol (96%). Dithizone Solution A 0.05% w/v solution in ehloroform.
Disodium Hydrogen Orthophosphate Disodium Prepare imrnediately before use.
hydrogen phosphate; Na2HP04,12H20 = 358.1 (10039- Dithizone Solution R2 Dissolve 40.0 mg of dithizone in
32-4) ehloroform and dilute to 1000.0 mL with the same solvent.
Disodium phosphate dodecahydrate of the British Dilute 30.0 mL ofthe solution to 100.0 mL with ehloroform.
Pharmacopoeia. Assay Dissolve a quantity of mereurie ehloride equivalent to
Disodium Hydrogen Orthophosphate, Anhydrous 0.1354 g of HgCI2 in a mixture of equal volumes of dilute
Anhydrous disodium hydrogen phosphate; suljurie acid and water and dilute to 100.0 mL with the same
Na2HP04 = 142.0 (7558-79-4) mixture of solvents. Dilute 2.0 mL of this solution to
Analytical reagent grade of commerce. 100.0 mL with a mixture of equal volumes of dilute sulfurie
Disodium Hydrogen Orthophosphate Dihydrate acid and water. (This solution contains 20 ppm of Hg).
Transfer 1.0 mL of the solution to a separating funnel and
Disodium hydrogen phosphate dihydrate;
add 50 mL of di/ute sulfurie acid, 140 mL of water and
Na2HP04,H20 = 178.0 (10028-24-7)
10 mL of a 200 gIL solution of hydroxylamine hydroehloride.
Disodium phosphate dihydrate of the British Pharmacopoeia. Titrate with the dithizone solution; after each addition, shake
Disodium Hydrogen Phosphate See Disodium hydrogen the mixture twenty times and towards the end of the titration
orthophosphate. aIlow to separate and discard the chloroform layer. Titrate
Disodium Hydrogen Phosphate, Anhydrous See until a bluish-green colour is obtained. Calculate the
Anhydrous disodium hydrogen orthophosphate. equivalent in micrograms of mercury per millilitre of the
Disodium Hydrogen Phosphate Dihydrate See dithizone solution from the expression 20/V, where Vis the
Disodium hydrogen orthophosphate dihydrate. volume in millilitres of the dithizone solution used in the
Disodium Hydrogen Phosphate Solution A 9% w/v titration.
solution. Divanadium Pentoxide Vanadium(v) oxide;
V20 5 = 181.9 (1314-62-1)
Disodium Tetraborate See Sodium tetraborate.
Ditalimphos O,O-Diethyl (1,3-dihydro-l,3-dioxo-2H- Content, minimum 98.5%.
isoindol-2-yl)phosphonothioate; C¡ 2H¡ 4N0 4PS = 299.3 YeIlow-brown or rust-brown powder, slightIy soluble in
(5131-24-8) water, soluble in strong mineral acids and in solutions of
alkali hydroxides with formation of salts.
Very slightIy soluble in water, in ethyl acetate and in
anhydrous ethano!. Appearance of solution Heat 1 g for 30 minutes with
10 mL of suljurie acid. AIlow to cool and dilute to 10 mL
A suitable certified reference solution may be used.
with the same acid. The solution is clear (2.2.1).
Sensitivity to hydrogen peroxide Dilute 1.0 mL of the Doxycycline Doxycyc1íne Monohydrate of the British
solution prepared for the test for appearance of solution Pharmacopoeia.
cautiously to 50.0 mL with water. To 0.5 mL of the solution n-Eicosane C ZO H 4Z = 282.6 (112-95-8)
add 0.1 mL of a solution of hydrogen peroxide (0.1 gIL of General reagent grade of commerce.
H zOz) . The solution has a distinct orange colour compared
with a blank prepared from 0.5 mL of the solution to be
examined and 0.1 mL of water. After the addition of 0.4 mL Echinacoside 2-(3,4-Dihydroxyphenyl)ethyl-3-0-(6-deoxy-
of hydrogen peroxide solution (0.1 gIL H zOz), the orange <X-L-mannopyranosyl)-4-0-[3-(3,4-
solution becomes orange-yellow. dihydroxyphenyl)propenoyl)-6-0- (~-o-glucopyranosyl)-~-D
Loss on ignition Maximum 1.0%, determíned on 1.00 g glycopyranoside; C3sH460Z0 = 786.5 (82854-37-3)
at 700 ± 50°. General reagent grade of commerce.
Assay Dissolve 0.200 g with heating in 20 mL of a 70% Edotreotide N-[[4,7, 10-Tris(carboxymethyl)-1,4, 7,1 0-
w/w solution of sulfuric acid. Add 100 mL of water and tetraazacyc1ododecan-l-yl] acetyl]-o-phenylalanyl-L-cysteínyl-
0.02 M potassium permanganate until a reddish colour is L-tyrosyl-o-tryptophyl-L-Iysyl-L-threonyl-N-[(IR,2R)-2-
obtained. Decolorise the excess of potassium permanganate hydroxy-l-(hydroxymethyl)propyl)-L-cysteínamide cyc1ic
by the addition of a 30 gIL solution of sodium nitrite. Add (2---+7)-disulfide, DOTATOC, DOTA-[Tyr3]-octreotide;
5 g of urea and 80 mL of a 70% w/w solution {)f sulfuric acid C6sHgzN1 401SSZ = 1422 (204318-14-9)
Coo!. U sing 0.1 mL of ferroin as indicator, titrate the Appearance, white or almost white powder.
solution immediately with 0.1 M ferrous sulfate until a Content, mínimum 95.0%.
greenish-red colour is obtained.
Electrolyte Reagent for the Determination of Water
1 mL of 0.1 M ferrous sulfate is equivalent to 9.095 mg of
Anhydrous reagent of commerce of combination of
V 2 0 S' anhydrous reagents for the coulometric determination of
Divanadium Pentoxide Solution in Sulfuric Acid water, containing suitable organic bases, sulfur dioxide and
Divanadium Pentoxide Solution in Sulphuric Acid iodide dissolved in a suitable solvent.
Dissolve 0.2 g of divanadium pentoxide in 4 mL of sulphuric Embonic Acid C23H1606 = 388.4 (130-85-8)
acid and dilute to 100 mL with water. General reagent grade of commerce.
Divinylbenzene and Vinylpyrrolidone Copolyrner for
Emetine Dihydrochloride
Chromatography
C2gH40Nz04,2HCI,5H20 = 643 .6 (316-42-7 (anhydrous))
Spherical partic1es (30 Jlm) of an m-divinylbenzene and N- Emodin 1,3,8-Trihydroxy-6-methylanthraquinone;
vínylpyrrolidone copolymer. C1sHlQOs = 270.2 (518-82-1)
Docosahexaenoic Acid MethyI Ester DHA methyl ester. General reagent grade of commerce.
Cervonic acid methyl estero (all-Z)-Docosa-4,7,10,13,16,19- Orange crystals; melting point, about 253°, with
hexaenoic acid methyl ester; C23H3402 = 342.5 (301-01-9) decomposition.
Content, mínimum 90.0%, determíned by gas Complies with the following test.
chromatography. Homogeneity Carry out test C for Identification described
Docusate Sodium See Dioctyl sodium sulfosuccinate. in the monograph for Rhubarb. The chromatogram obtained
Dodecan-l-ol See Lauryl alcohol with solution (2) shows only one principal spo t.
4-DodecylresorcinoI CH3(CH z) 11 C6Hr 1,3-(OH)z Endoprotease LysC Microbial extracellular proteolytic
= 278.43 (24305-56-4) enzyme secreted by Achromobacter lyticus. A Iyophilised
General reagent grade of commerce. powder, free of salts.
<x-Endosulfan o:-Endosulphan;
Dodecyltrimethylammonium Bromide
CgH 6Cl 60 3S = 406.9 (959-98-8)
C 1sH 34NBr = 308.4 (1119-94-4)
Use a grade of commerce suitable for pesticide residue
White or almost white crystals.
analysis. A suitable certified reference solution (10 ng/JlL in
Melting point, about 246 oC. cyc1ohexane) may be used.
Domiphen Bromide Dodecyldimethyl-2- Boiling point, about 200°; melting point, about 108°.
phenoxyethylammonium bromide; C 22 H 40 BrNO = 414.5 ~-Endosulfan ~-Endosulphan;
(538-71-6) CgH 6Cl 60 3S = 406.9 (33213-65-9)
General reagent grade of commerce. Use a grade of commerce suitable for pesticide residue
0- Dopa (2R)-2-Amino- 3-(3,4-dihydroxyphenyl)propanoic analysis. A suitable certified reference solution (10 ng/JlL in
acid, 3-hydroxy-o-tyrosine, 3,4-dihydroxy-o-phenylalanine; cyc1ohexane) may be used.
CgH l1 N0 4 = 197.2 (5796-17-8) Boiling point, about 390°; melting point, about 207°.
[a]ijl + 9.5 to + 11.5, determined on a 10 gIL solution in A suitable certified reference solution (10 ng/JlL in
1 M hydrochloric acid; melting point, about 277°. cyc1ohexane) may be used.
Dotriacontane C32H66 = 450.9 (544-85-4) Endrin C 1zH s CI 60 = 380.9 (72-20-8)
White or almost white plates, practically insoluble in water, Use a grade of commerce suitable for pesticide residue
sparingly soluble in hexane. analysis. A suitable cenified reference solution (10 ng/JlL in
Melting point, about 69°. cyclohexane) may be used.
Impurities Not more than 0.1 % of impurities with the Eosin CI 45380; acid red 87;
same tR value as <x-tocopherol aceta te, determined by the gas C2oH6Br4Na20S = 691.9 (17372-87-1)
chromatographic method prescribed in the monograph General reagent grade of commerce.
Alpha-Tocopherol acetate (0439) . A red powder.
Erucamide (Z)- Docos-13-enoarnide; ehromatography, Appendix III B, altemately injecting 1 !lL of

C 22 H 43NO = 337.6 (112-84-5) the following solutions three times. For solution (1) use the
General reagent grade of commerce. reagent being examined. For solution (2) dilute 0.5 mL of
anhydrous methanol to 100 mL with the reagent being
exarnined and further dilute 1 mL to 100 mL.
Erythritol meso-Erythritol; (R*,S")-butane-1,2,3,4-tetrol;
The chromatographic procedure may be carried out using a
C 4H lO 0 4 = 122.1 (149-32-6)
glass column (2 m x 2 mm) packed with ethylvinylbenzene-
General reagent grade of commerce. divinyl benzene copolymer (75 !lm to 100 !lm) and using
Melting point, about 12l.5°. 30 mL per minute as the flow rate of the carrier gas.
Esculin ClsH1609,1 'hH 20 = 367.3 (531-75-9) Maintain the temperature of the column at 130°, that of the
General reagent grade of commerce complying with the injection port at 150° and that ofthe detector at 200°. After
following test. each injection heat the colurnn to 230° for 8 minutes.
Chromatographic purity Carry out Identification test C Calculate the percentage of methanol using the expression
in the monograph for Eleutherococcus using a 0.020% w/v ab/(e - b) where a is the percentage v/v content of methanol
solution of the reagent being examined in a mixture of in solution (2), bis the area of the methanol peak in the
2 volumes of warer and 8 volurnes of ethanol (50%). The chromatogram obtained with solution (1) and e is the area of
chromatogram shows onIy one principal spot. the methanol peak in the chromatogram obtained with
solution (2).
Estradiol Estra-1,3,5(lO)-triene-3,17~-diol. ~-Estradiol;
ClsH2402 = 272.4 (50-28-2) Ethanol (96%) Alcohol
Prisms stable in air, practically insoluble in water, freely Analytical reagent grade ethanol of commerce containing not
soluble in alcohol, soluble in acetone and in dioxane, les s than 95.1 % v/v and not more than 96.9% v/v of C 2H 6 0 .
sparingly soluble in vegetable oils. A colourless liquid; weight per mL, about 0.81 g.
Melting point, 173° to 179°. Dihlted ethanols may be prepared by diluting the volumes of
17cx-Estradiol C 1s H 24 0 = 272.4 (57-91-0) ethanol (96%) indicated in the following table to 1000 mL
with water.
Estragole 4-AlIylanisole; 1-methoxy-4-prop-2-enylbenzene;
C lOH l2 0 = 148.2 (140-67-0) Strength Volume of emanol Weight per
General reagent grade of commerce. (96%) (approx) mL
% v/v rnL g
Boiling point, about 216°; n~, about l.52.
90 934 0.83
Estragole used in gas ehromatography complies with the following 85 885 0.85
test. 80 831 0.86
Assay Examine by gas ehromatography, Appendix 111 B, 70 727 0.89
under the conditions described in the test for 65 676 0.90
Chromatographic profile in the monograph for Anise Oil 60 623 0.91
using the reagent being examined as the test solution. 50 519 0.93
The area of the principal peak is not les s than 98.0% of the 45 468 0.94
total area of the peaks. 25 259 0.97
Ethane-l,2-diol Ethylene glycol; C 2H 6 0 2 = 62.07 (107- 20 207 0.975
21-1) 10 104 0.986
A colourless, viscous liquid; d~g, 1.113 to l.115; n~,
Ethanol (96%), Aldehyde-free Aldehyde-free alcohol
l.430 to l.433; boiling point, about 196°.
Mix 1200 mL of ethanol (96%) with 5 mL of a 40% w/v
Acidity T o 10 mL add 20 mL of water and 1 mL of
solution of silver nitrate and 10 mL of a cooled 50% w/v
phenolphthalein solution. Not more than 0.15 mL of 0.02 M
solution of potassium hydroxide. Shake, allow to stand for a
sodium hydroxide is required to change the colour of the
few days and filter. Distil the filtrate immediately before use.
indicator to pink.
Ethanolarnine 2-Aminoethanol; C 2H 7 NO = 61.08 (141-
Water Not more than 0.2%.
43-5)
Ethanol See Absolute ethanol.
Ethanol, Absolute Ethanol; C 2H 60 = 46.07 (64-17-5)
A colourless, viscous liquid; d~g, about l.04; n~, about
Analytical reagent grade of commerce containing not less l.454; melting point, about 11 c .
than 99.5% v/v of C 2H 60.
A colourless, hygroscopic liquid; boiling point, 78° to 79°;
Ether Diethyl ether; C 4H IO O = 74.12 (60-29-7)
d~g, 0.791 to 0.794.
Store protected from light at a temperature not exceeding
30°. A volatile, highly flarnmable, colourless liquid; boiling point,
34° to 35°; d~g, 0.713 to 0.715.
Ethanol Rl See Absolute ethanol R1 .
Do not distil unless the ether complies with the following test
Ethanol Rl, Absolute Ethanol R1
for peroxides.
Absolute ethanol grade of commerce complying with the
Peroxides Place 8 mL of potassium iodide and stareh
following test.
solution in a 12 mL ground-glass-stoppered cylinder about
Methanol Not more than 0.005% v/v when determined in 1.5 cm in diameter. Fill completely with the reagent being
the following manner. Carry out the method for gas
examined, shake vigorously and allow to stand in the dark A colourless or almost colourless liquid; boiling point, 205 0
for 30 minutes. No colour is produced. to 2090, with decomposition.
Store protected from light at a temperature not exceeding Ethyl Formate C 3 H 60 2 = 74. 1 (109-94-4)
15°. The name and concentration of any added stabiliser are General reagent grade of commerce.
stated on the labe!'
d~g, about 0.919; n~o, about 1.36; boiling point, about 54°.
Ether, Peroxide-free Shake 1000 mL of ether with
Ethyl 4-Hydroxybenzoate See Ethyl parahydroxybenzoate.
20 mL of a solution of 30 g of iron(Il) sulfate in 55 mL of
water and 3 mL of sulfuric acid. Continue shaking until a Ethyl Methanesulfonate C 3 H s0 3 S = 124.2 (62-50-0)
small sample no longer produces a blue colour when shaken Clear, colourless liquido
with an equal volume of a 2% w/v solution of potassium iodide Content, minimum 99.0%; density, about 1.206 g cm- 3
and 0.1 mL of starch mucilage. (20°).
Ethion C9H2204PZS4 = 384.5 (563-12-2) n~o, about 1.418; boiling point, about 213°.
Use a grade of commerce suitable for pesticide residue Ethyl Methyl Ketone See Butan-2-one.
analysis. A suitable certified reference solution (10 ng/flL in Ethyl Parahydroxybenzoate Ethyl 4-hydroxybenzoate;
cyclohexane) may be used. C 9 H IO 0 3 = 166.2 (120-47-8)
Melting point, - 24° to -25°. General reagent grade of commerce.
Ethoxychrysoidine Hydrochloride 4-p-Ethoxyphenylazo- Melting point, about 117 0 •
m-phenylenediamine hydrochloride;
4-[ (Ethylamino )methyl]pyridine
C¡4H¡6N40,HCI = 292.8 (2313-87-3)
C sH 12N 2 = 136.2 (33403-97-3)
A red powder.
d~g, about 0.98; n~, about 1.516; boiling point, about 98°.
Ethoxychrysoidine Solution A 0.1 % w/v solution of
Ethylbenzene CsH IO = 106.2 (100-41-4)
ethoxychrysoidine hydrochloride in ethanol (96%).
Reagent grade of commerce containing not less than 99.5%
w/w of CSH IO when determined by gas chromatography.
Sensitivity to bromine To a mixture of 0.05 mL and
A colourless liquid; d~g, about 0.87; n~, about 1.496; boiling
5 mL of 2M hydrochloric acid add 0.05 mL of
point, about 135°.
0.0167M bromine Vs. The colour changes from red to light
yellow within 2 minutes. 4-Ethylcatechol 3,4-Dihydroxyethylbenzene;
C SH IO 0 2 = 138.2
2-Ethoxyethanol Ethylene glycol monoethyl ether;
C 4H IO 0 2 = 90.1 (110-80-5) General reagent grade of comrnerce.
General reagent grade of commerce. Ethylene Bis [3 ,3-di (3 - (1, l-dimethyl) ethyl-4-
hydroxyphenyl)butyrate] C50H660S = 795 (325 09-66-3)
Boiling point, about 135°; d~g, about 0.93; n~o, about 1.406.
Ethyl Acetate C 4H s 0 2 = 88.1 (141-78-6)
Ethylene Bis[3,3-di(3-tert-butyl-4-
A colourless liquid; boiling point, about 76° to 78°; d~g,
hydroxyphenyl)butyrate] See Ethylene bis[3,3-di(3-(1,1-
0.901 to 0.904.
dimethyl) ethyl-4-hydroxyphenyl) butyrateJ.
Ethyl Acetate, Treated Disperse 200 g of sulfamic acid in
Ethylene Chloride See 1,2-Dichloroethane.
sufficient ethyl acetate to produce 1000 mL, stir the
suspension for 3 days and filter. Ethylene Glycol See Ethane-1,2-diol.
Use within 1 month of preparation. Ethylene Glycol Monoethyl Ether 2-Ethoxyethanol;
C 4 H IO 0 2 = 90.1 (110-80-5)
Ethyl Acrylate Ethyl prop-2-enoate;
C 5H s 0 2 = 100.1 (140-88-5) Content, minimum 99.0%.
General reagent grade of commerce. A clear, colourless liquid, miscible with water, with acetone
and with ethanol (96%).
Melting point, about -71 °; boiling point, about 99°; n~o,
about 1.406; d~g, about 1.04. d~g, about 0.93; boiling point, about 135°.
Ethyl Benzoate C 9 H IO 0 2 = 150.2 (93-89-0) Ethylene Glycol Monomethyl Ether 2-Methoxyethanol;

C 3 H s 0 2 = 76.1 (109-86-4)
A colourless liquid; n~, about 1.505; weight per mL, about
1.05 g. A clear, colourless liquid, miscible with water, with acetone
and with ethanol (96%).
Ethyl 5-Bromovalerate Ethyl 5-bromopentanoate;
C7H I3Br02 = 209.1 (14660-52-7) d~g, about 0.97; n~o, about 1.403; boiling point, about 125°.
Clear, colourless liquido Ethylene Oxide Oxirane; C 2 H 40 = 44.05 (75-21-8)

d~g, about 1.321 ; boiling point, 104° to 109°. General reagent grade of commerce.
Ethyl Cinnamate C¡¡HI 20 Z = 176.2 (103-36-6) A colourless gas; liquefaction point, about 12°.
General reagent grade of commerce. Ethylene Oxide Solution Weigh a quantity of cool
ethylene oxide stock solution containing 2.5 mg of ethylene
A colourless or very pale yellow liquid; weight per mL, about
oxide into a cool fiask and dilute to 50 g with polyethylene
1.05 g.
glycol200 R1 . Mix well and dilute 2.5 mg ofthis solution to
Ethyl Cyanoacetate C 5H 7N0 2 = 113.1 (105-56-6) 25 mL with polyethylene glycol 200 R1 (5 flg of ethylene oxide
Analytical reagent grade of commerce. per g of solution).
Prepare immediately before use .
Ethylene Oxide Solution Rl Dilute 1 mL of cooled Boiling point, about 116 0 ; n~o , about 1.457; weight per mL,
ethylene oxide stock solunon (check the exact volume by about 0.90 g.
weighing) to 50 mL with polyethylene glycol 200 R1 and mix Ethylenediaminetetra-acetic Acid
well. Dilute 2.5 mg of this solution to 25 mL with ClQHI 6N ZOS = 292.2 (60-00-4)
polyethylene glycol 200 R1 (5 [!g of ethylene oxide per g of General reagent grade of cornmerce.
solution) . Calculate the exact amount of ethylene oxide in
ppm from the volume determined by weighing and taking Melting point, about 250°.
the density of polyethylene glycol 200 R1 to be 1.127. (Ethylenedinitrilo) tetra-acede Acid
See Ethylenediaminetetra-acetic acid.
Ethylene Oxide Solution R2 Prepare immediately before 2-EthyIhexane-l,3-diol C SH 180 Z = 146.2 (94-96-2)
use. Weigh 1 g of cold ethylene oxide stock solunon (equivalent General reagent grade of commerce.
to 2.5 mg of ethylene oxide) into a cold f1ask containing dig, about 0.942; n~o, about 1.451; boiling point, about
40.0 g of cold polyethylene glycol 200 R1. Mix and determine 244 0 •
the exact weight and dilute to a calculated weight to obtain a 2-Ethylhexanoic Acid 2-Ethylhexoic acid;
solution containing 50 [!g of ethylene oxide per g of solution. C SH I60 2 = 144.2 (149-57-5)
Weigh 10 g into a f1ask containing about 30 mL of water, General reagent grade of commerce.
mix and dilute to 50 mL with water (lO ~lg per rnL of
ethylene oxide). A colourless liquid; dig, about 0.91; n~, about 1.425.
Ethylene Oxide Solution R3 Prepare immediately before Complies with the following test.
use. Dilute 10 mL of ethylene oxide solunon R2 to 50 mL with Related substances Carry out the method for gas
water (2 [!g per mL of ethylene oxide) . chromatography, Appendix III B, using 1 [!L of a solution
Ethylene Oxide Solution R4 Dilute 1.0 mL of ethylene prepared in the following manner. Suspend 0.2 g of the
oxide stock solution R1 to 100.0 mL with water. Dilute reagent in 5 mL of water, add 3 rnL of 2M hydrochlO1ic acid
1.0 mL of this solution to 25.0 mL with water. and 5 mL of hexane, shake for 1 minute, allow the layers to
separate and use the upper layer.
Ethylene Oxide Solution R5 A 50 gIL solution of ethylene
oxide R in methylene chloride R. The chromatographic procedure described in the test for 2-
Ethylhexanoic acid in the monograph for Amoxicillin Sodium
Either use a commercially available reagent or prepare the may be used.
solution corresponding to the above-mentioned composition.
The sum of the areas of any secondary peaks is not greater
Ethylene Oxide Stock Solution Al! operations carried out than 2.5% of the area of the principal peak.
in the preparanon of these solutions must be conducted in a fume-
hood. The operator must protect both hands and face by wearing l,l'-Ethylidenebis(tryptophan)
polyethylene protective gloves and an appropriate face mask. Store C24H26N404 = 434.5 (132685-02-0)
all solunons in airtight containers at 4 to 8". Carry out all
0
determinanons three times. 98.0% of C24H 26N404'
Into a dry, c1ean test tube cooled in a mixture of 1 part of Melting point, about 223 with decomposition.
0
,
sodium chloride and 3 parts of crushed ice introduce a slow Assay Proceed as described in the test for 1,1'-
current of ethylene oxide gas, allowing condensation onto the Ethylidenebis(tryptophan) and other related substances in
inner wall of the test tube. Using a glass syringe, previously the monograph for Tryptophan. The area of the principal
cooled to -100, inject about 300 [!L (corresponding to about peak in the chromatogram obtained with reference solution
0.25 g) of liquid ethylene oxide into 50 mL of polyethylene (a) is not les s than 98.0% of the area of all the peaks.
glycol 200 R1. Determine the absorbed quantity of ethylene N- Ethylrnaleimide 1-Ethyl-lH-pyrrole-2,5-dione;
oxide by weighing before and after absorption (MEO)' Dilute C 6 H 7N0 2 ~ 125.1 (128-53-0)
to 100 mL with polyethylene glycol 200 R1. Mix well before General reagent grade of commerce.
use. Determine the exact concentration of ethylene oxide in
the following manner. Melting point, 41 0 to 45 0 •
Assay To 10 mL of 50% w/v suspension of magnesium Store at a temperature between 20 and 8 0 •
chloride in absolute ethanol add 20 mL of O.l M ethanolic 2-Ethyl-2-methylsuccinic Acid 2-Ethyl-2-
hydrochloric acid VS, shake to obtain a saturated solution and methylbutanedioic acid; C 7H 120 4 = 160.2 (631-31-2)
allow to stand ovemight to equilibrate. Weigh 5 g of the General reagent grade of commerce.
solution being examined into the f1ask and allow to stand for Melting point, 104 0 to 107°.
30 minutes. Titrate with O.lM ethanolic potassium hydroxide
2-Ethylpyridine C7H9N = 107.2 (100-71-0)
VS, determining the end point potentiometrically,
Appendix VIII B. Carry out a blank titration using General reagent grade of commerce.
polyethylene glycol 200 R1 in place of the substance being n~o, about 1.496; dig, about 0.939; boiling point, about
examined. The ethylene oxide concentration in mg per g is 149 0 •
given by the expression 4.404(Vo - V1) /m, where Vo and VI l-Ethylquinaldinium Iodide l-Ethyl-2-
are the volumes of ethanolic potassium hydroxide used methylquinolinium iodide; C 12H 14IN = 299.2 (606-55-3)
respectively for the blank titration and the assay and m is the General reagent grade of commerce.
weight of sample taken, in g.
Ethylvinylbenzene-Divinylbenzene Copolymer
Ethylene Oxide Stock Solution Rl A 50 mg!mL solution
Porous, rigid, cross-linked polymer beads.
of ethylene oxide R in methanol R.
Gas chromatographic grade of commerce.
Ethylenediamine 1,2-Diaminoethane;
CzHsNz = 60.10 (107-15-3) Several grades are available with different sizes of bead; the
size range is specified after the name of the reagent in tests
where it is used.
Ethylvinylbenzene-Divinylbenzene Copolymer Rl Euglobulins, Human

Porous, rigid, cross-linked polymer beads with a nominal For the preparation, use fresh human blood collected into an
specific surface area of 500 m 2/g to 600 mZ/g and having anticoagulant solution (for example sodium citrate solution)
pores with a mean diameter of 7.5 nm. or human blood for transfusion collected into plastic blood
Gas chromatographic grade of commerce. bags that has just reached its expiry date. Discard any
Several grades are available with different sizes of bead; the haemolysed blood. Centrifuge at 1500 to 1800 g at 15° to
size range is specified after the name of the reagent in tests obtain a supernatant plasma poor in platelets. Iso-group
where it is used. plasmas may be mixed.
Eugenol 4-Allyl-2-methoxyphenol; To 1 litre of the human plasma add 75 g of banum sulfate
C lO H 12 0 Z = 164.2 (97-53-0) and shake for 30 minutes. Centrifuge at not les s than 15,000
General reagent grade of commerce. g at 15° and draw off the clear supernatant liquido
Add 10 mL of a 0.02% w/v solution of aprotinin and shake to
A colourless or pale yellow, oily liquid; dig, about l.07. ensure mixing. In a container with a minimum capacity of
Eugenol used in gas ehromarography eomplies with the following 30 litres in a chamber at 4° introduce 25 litres of distilled
test. water at 4° and add about 500 g of solid carbon dioxide.
Assay Carry out the test for Chromatographic profile in Irnmediately add, while stirring, the supernatant liquid
the monograph for Clove Oil using the reagent being obtained from the plasma. A white precipitate is produced.
examined as the test solution. The area of the principal peak Allow to settle at 4° for 10 to 15 hours. Remove the clear
is not less than 98.0% of the total area of the peaks. supernatant solution by siphoning. Collect the precipitate by
Store protected from light. centrifuging at 4°. Suspend the precipitate by dispersing
mechanically in 500 mL of distilled water at 4°, shake for
Euglobulins, Bovine
5 minutes and collect the precipitate by centrifuging at 4°.
For the preparation, use fresh bovine blood collected into an Disperse the precipitate mechanically in 60 mL of a solution
anticoagulant solution (for example sodium citrate solution). containing 0.9% w/v of sodium ehlonde and 0.09% w/v of
Discard any haemolysed blood . Centrifuge at 1500 to 1800 g sodium citrate and adjust the pH to 7.2 to 7.4 by adding a
between 15° and 20° to obtain a supernatant plasma poor in 1% w/v solution of sodium hydroxide. Filter through a
platelets. sintered-glass filter; to facilitate the dissolution of the
To 1 litre of the bovine plasma add 75 g of banum sulfate precipitate crush the particles of the precipitate with a
and shake for 30 minutes. Centrifuge at not less than 1500 g suitable implemento Wash the filter and the implement with
to 1800 g between 15° and 20° and draw off the clear 40 mL of the ch1oride-citrate solution described aboye and
supernatant liquido Add 10 mL of a solution of aprotinin dilute to 100 mL with the same solution. Freeze dry the
containing 0.2 mg per mL and shake to ensure mixing. In a solution. The yields are generally 6 to 8 g of euglobulins per
container with a minimum capacity of 30 litres in a chamber litre of human plasma.
at 4° introduce 25 litres of distilled water at 4° and add about Complies with the following test.
500 g of solid carbon dioxide. Immediately add, while
stirring, the supernatant liquid obtained from the plasma. A Suitability For this test, prepare the solutions using
white precipitate is produced. Allow to settle at 4° for 10 to phosphate buffer solution pH 7.2 containing 3.0% w/v of bovine
15 hours. Remove the clear supernatant solution by albumino Into a test tube 8 mm in diameter placed in a
siphoning. Collect the precipitate by centrifugation at 4°. water-bath at 37° introduce 0.1 mL of a solution of a
Suspend the precipitate by dispersing mechanically in reference preparation of streptokinase containing 10 IU of
500 mL of distilled water at 4°, shake for 5 minutes and streptokinase activity per millilitre and 0.1 mL of a solution
collect the precipitate by centrifugation at 4°. Disperse the of human thrombin containing 10 IU per millilitre.
precipitate mechanically in 60 mL of a solution containing Add rapidly 1 mL of a solution containing 10 mg of human
0.9% w/v of sodiwn ehloride and 0.09% w/v of sodium eitrate euglobulins per millilitre. A firrn clot forrns in less than
and adjust the pH to 7.2 to 7.4 by adding a 1% w/v solution 10 seconds. Note the time that elapses between the addition
of sodium hydroxide. Filter through a sintered-glass filter; of the solution of human euglobulins and the lysis of the
to facilitate the dissolution of the precipitate crush the clot. The lysis time does not exceed 15 minutes.
partic1es of the precipitate with a suitable implemento Wash Store in an airtight container at 4° and use within 1 year.
the filter and the implement with 40 mL of the ch1oride- (E,E)-Famesol trans,trans-Famesol; (2E,6E)-3,7,11-
citrate solution described aboye and dilute to 100 mL with Trimethyldodeca-2,6, 1O-trien-l-ol;
the same solution. Freeze dry the solution. The yields are C 1sH 26 0 = 222.4 (106-28-5)
generally 6 to 8 g of euglobulins per litre of bovine plasma. Fast Blue B Salt CI37235;
Suitability test For this test, prepare the solutions using C14H12Cl2N402 = 339.2 (84633-94-3)
phosphate buffer pH 7.4 containing 3% w/v of bovine albumino General reagent grade of commerce.
Into a test tube 8 mm in diameter placed in a water bath at
A dark green powder, stabilised by the addition of zinc
3r, introduce 0.2 mL of a solution of a reference chloride.
preparation of urokinase containing 100 IU of urokinase
activity per mL and 0.1 mL of a solution of thrombin Store in an airtight container at a temperature between 2°
containing 20 IU per mL. Add rapidly 0.5 mL of a solution and 8°.
containing 10 mg of the euglobulin fraction per mL. A firrn Fast Red B Salt CI 37125; 2-methoxy-4-
clot is produced in less than 10 seconds. Note the time that nitrobenzenediazonium hydrogen naphthalene-l,5-
elapses between the addition of the solution of the disulfonate; C 17H\3N3 0 9S 2 = 467.4 (49735-71-9)
euglobulin fraction and the lysis of the clot. The lysis time Orange-yellow powder, soluble in water, slightly soluble in
does not exceed 15 minutes. ethanol (96%) .
Store protected from moisture at 4° and use within 1 year. Store in an airtight container protected from light at a
temperature of 2° to 8°.
Fenbufen Cl6Hl403 = 254.3 (36330-85-5) Ferroin Solution (14634-91-4)

General reagent grade of commerce. Tris(I,IO-phenanthroline)iron(n) sulfate complex prepared in
Fenchlorphos C sH sCl 30 3PS = 321.5 (299-84-3) the following manner. Dissolve 0.7 g of iron(lI) sulfate and
1.76 g of phenanthroline hydrochloride in 7 O mL of water and
add sufficient water to produce 100 mL.
analysis. A suitable certified reference solution (lO ng/JlL in
cyclohexane) may be used. Complies with the following test.
Melting point, about 35°. Sensitivity to cerium(IV) Add 0.1 mL ofthe solution and
0.15 mL of osmium tetroxide solution lO 50 mL of 1M sulfurie
Fenchone (lR)-1,3,3-Trimethylbicyclo[2.2.1]heptan-2-
acid. Add 0.1 mL of O.IM ammonium cerium(IV) nitrate. The
one; C IOH l60 = 152.2 (7787-20-4)
colour changes from red to light blue.
Oily liquid, miscible with ethanol (96%), practically insoluble
Ferrous Ammonium Sulfate See Ammonium iron(lI)
in water.
sulfate.
Boiling point, 192° to 194°; n5°, about 1.46.
Ferrous Sulfate See Iron(lI) sulfate.
Fenchone used in gas chromawgraphy complies with the following
Ferrous Sulfate Solution R2 See Iron(lI) sulfate
test.
solution R2.
Assay Examine by gas chromawgraphy, Appendix III B,
Ferulic Acid 4-Hydroxy-3-methoxycinnamic acid; 3-
under the conditions described in the monograph for Bitter
(4-hydroxy-3-methoxyphenyl)propenoic acid;
Fennel using the reagent being examined as the test solution.
C 1oH IO 0 4 = 194.2 (1135-24-6)
The area of the principal peak is not less than 98.0% of the
Faint yellow powder, melting point, 172.9° to 173.9°.
total area of the peaks.
Ferulic acid used in the assay of eleutherosides in
Fenvalerate C2sH22CIN03 = 419.9 (51630-58-1)
Eleutherococcus complies with the following test.
Assay Examine by liquid chromalOgraphy (2.2.29) as
analysis, A suitable certified reference solution (lO ng/JlL in
prescribed in the monograph on Eleutherococcus.
cyclohexane) may be used.
The content is not les s than 99%, calculated by the
normalisation procedure.
Ferric Ammoniurn Sulfate See Ammonium iron(m)
Fibrin Blue Mix 1.5 g of fibrin with 30 mL of a 0.5% w/v
sulfate.
solution of indigo carmine in 0.02M hydrochloric acid. Heat the
Ferric Ammoniurn Sulfate Solution R2 See Ammonium mixture to 80° and maintain at this temperature while
iron (m) solution R2. stirring for about 30 minutes. Allow lO cool, filter and wash
Ferric Ammoniurn Sulfate Solution R5 See Ammonium e.xtensively by resuspension in 0.02M hydrochlon'c acid and
iron(m) solution R5. mixing for about 30 minutes. Filter and repeat the washing
Ferric Ammoniurn Sulfate Solution R6 See Ammonium procedure three time. Dry the residue at 50° and grind.
iron(m) solution R6. Fibrin Congo Red See Congo red fibrin.
Ferric Chloride See lron(m) chloride. Fibrinogen (9001-32-5)
Ferric Chloride Solution Rl See lron(m) chloride Complies with the monograph for Dried Fibrinogen.
solution R 1. Fixing Solution To 250 mL of methanol add 0.27 mL of
Ferric Chloride Solution R2 See lron(m) chloride formaldehyde solution and sufficient water to produce 500 mL.
solution R2 Fixing Solution for Isoelectric Focusing in
Ferric Chloride Solution R3 A 2.0% w/v solution of Polyacrylamide Gel
iron (m) ehloride hexahydrate in absolute ethanol. A solution containing 35 g of sulfosalicylic acid and lOO g of
Ferric Chloride Solution, RadiolabelIed [ 59Fe], trichloroacetic acid per litre of water.
Concentrated A commercially available solution of Flufenamic Acid Cl 4HIOF3N02 = 281.2 (53 0-78-9)
[s9Fe]ferric chloride (approximate specific activity:
Pale yellow, crystalline powder or needles.
100-1000 MBq/mg of Fe).
Ferric Chloride Solution, RadiolabelIed [ 59Fe] Dilute
the concentrated radiolabelled 19Pelferric chloride solution in Melting point, 132° lO 135°.
sodium citrate buffer solution pH 7.8 to obtain a solution with Flumazenil (78755-81-4) Of the British Pharmacopoeia.
an activity of 3.7 x 104 Bq/mL Flunitrazepam (1622-62-4) Of the British Pharmacopoeia.
Ferric Chloride-Sulfamic Acid Reagent See lron(m) Fluoranthene C l6 HIO = 202.3 (206-44-0)
chloride-sulfamic acid reagent. General reagent grade of commerce.
Ferric Nitrate See lron(m) nitrate. Melting point, 105° to 110°; boiling point, about 384°.
Ferric Sulfate See lron(m) sulfate. Fluorene Diphenylenemethane; C l3HIO = 166.2 (86-73-7)
Ferrocyphen Dicyanobis(I,IO-phenanthroline)iron(n); General reagent grade of cornmerce.
C26Hl6FeN6 = 468.3 (14768-11-7)
Melting point, 113° lO 115°.
A violet-bronze, crystalline powder.
9-Fluorenone C 13 H 1S O = 180.2 (486-25-9)
Store protected from light and moisture.
Ferrocyphen Solution Dissolve, without warming, 0.5 g
Fluorenone Solution Dissolve 50 mg of 9-fiuorenone in
of ferrocyphen in 50 mL of sulfuric acid.
10 mL of warm methanol, transfer lO a 500-mL graduated
Ferrocyphene See Ferrocyphen. fiask with the aid of l 90 mL of methanol and add sufficient
Ferroin See Ferroin solution. water lO produce 500 mL. The solution should be clear.
Immediately before use dilute 10 mL of this solution to 1M sodium hydroxide. AIIow to stand for 5 minutes, add
100 mL with methanol. 11 mL of 1M hydrochloric acid and titrate the excess iodine
(9-Fluorenyl)methyl Chloroformate Fluoren-9-ylmethyl with O.IM sodium thiosulfate VS using 1 mL of starch solution
chloromethanoate; C1sH Il CIO z = 258.7 (28920-43-6) added towards the end of the titration as indicator. Each mL
of 0.05M iodine VS is equivalent to 1.50 mg of CHzO.
Store at a temperature between 15° and 25°.
Formamide CH 3NO = 45.0 (75-12-7)
Fluorescamine C 17H IO 0 4 = 278.3 (38183-12-9)
A cIear, colourIess, oily Iiquid, hygroscopic, miscible with
water and with ethanol (96%). It is hydrolysed by water.
dig, about 1.134; boiling point, about 210°.
Fluorescein C2oHl20S = 332.3 (2321-07-5)
An orange-red powder displaying a green fluorescence in
solution.
Formamide Rl Formamide complying with the foIlowing
test.
Water Not more than 0.1% determined with an equal
Fluorescein Sodium Sodium fluoresceinate; volume of anhydrous methanol.
CZOHIONazOs = 376.3 (518-47-8)
Formamide, Treated Disperse 1 g of sulfamic acid in
General reagent grade of commerce. 20 mL ofjormamide containing 5% v/v of water.
2-Fluoro-2-deoXY-D-glucose Formic Acid CHzOz = 46.03 (64-18-6)
C 6HllFO s = 182.2 (86783-82-6)
Analytical reagent grade of commerce containing about 90%
General reagent grade of commerce. w/w of CHzOz and about 23.6M in strength .
Melting point, 174° to 176°. A colourIess, corrosive Iiquid; weight per mL, about 1.20 g.
2-Fluoro-2-deoxy-n-mannose Formic Acid, Anhydrous CHzOz = 46.03
C 6HllFO s = 182.1 (38440-79-8)
Analytical reagent grade formic acid of commerce containing
ColourIess semi-solid. not less than 98.0% w/w of CHzOz.
FIuorodinitrobenzene See 1-Pluoro-2,4-dinitrobenzene. A colourIess, corrosive liquid; dig, about 1.22.
1-FIuoro-2,4-dinitrobenzene 2,4-Dinitrofluorobenzene; Assay Weigh accurately a conical flask containing 10 mL
C6H3FNz04 = 186.1 (70-34-8) of water, quickIy add about 1 mL of the reagent and weigh
General reagent grade of commerce. again. Add 50 mL of water and titrate with 1M sodium
Pale yeIlow, vesicatory crystals, lumps or liquid with a hydroxide VS using 0.5 mL of phenolphthalein solution as
lachrymatory vapour. indicator. Each mL of 1M sodium hydroxide VS is equivalent
Melting point, about 29°; weight per mL, about 1.48 g; n~, to 46.03 mg of CHzOz.
about 1.569. Formononetin 7-Hydroxy-3-( 4-methoxyphenyl)-4-
nL-6-Fluorodopa Hydrochloride (2RS)-2-Amino-3- benzopyrone; 7-Hydroxy-3-(4-methoxyphenyl)chromone;
(2-fluoro-4,5-dihydroxyphenyl)propanoic acid hydrochloride; 7-Hydroxy-4 '-methoxyisoflavone; Cl6HI Z04
2-Fluoro-5-hydroxY-DL-tyrosine hydrochloride; = 268 .26 (485-72-3)
C 9H Il CIFN0 4 = 251.6 (1169200) General reagent grade of commerce.
White or almost white powder. Fructose See D-Fructose.
6-FIuorolevodopa Hydrochloride . (2S)-2-Amino-3- n-Fructose Laevulose; C 6H 1Z0 6 = 180.2 (57-48-7)
(2-fluoro-4,5-dihydroxyphenyl)propanoic acid hydrochloride, General reagent grade of commerce.
2-Fluoro-5-hydroXY-L-tyrosine hydrochloride; A white, crystaIline powder; melting point, about 103°, with
C 9H ll CIFN0 4 = 251.6 (144334-59-8) decomposition; [al~o, about - 92° (10% w/v in water
ColourIess or almost colourIess solid, soluble in water. containing 0.05 mL of 5M ammonia).
l-Fluoro-2-nitro-4-trifluoromethylbenzene 4-Fluoro-3- Fuchsin, Basic Basic violet 14; basic magenta; a mixture
nitrobenzotrifluoride; C7H3F4NOz = 209.1 (367-86-2) ofrosaniline hydrochloride, CZOHI 9N3,HCI (337.9) (CI
General reagent grade of commerce. 42510) and pararosaniline hydrochloride, C I9H I7N 3,HCI
Melting point, about 197°. (323.8) (CI 42500) (569-61-9)
Folie Acid Cl 9Hl9N706 = 441.4 (75708-92-8) A dark red powder or green crystals with a metaIlic lustre of
such a purity that, when used in the preparation of decolorised
juchsin solution, an almost colourIess solution is obtained. If
A yeIlow to orange, crystaIline powder. necessary, purify in the foIlowing manner. Dissolve 1 g in
Store in a weIl-cIosed container protected from light. 250 mL of 2M hydrochloric acid, aIlow to stand for 2 hours at
Formaldehyde See Formaldehyde solution. room temperature, filter and neutralise with 2M sodium
Formaldehyde Solution Formaldehyde; hydroxide and add 1 mL to 2 mL in excess. Filter the
CHzO = 30.03 (50-00-0) precipitate through a sintered-glass filter (40) and wash with
water. Dissolve the precipitate in 70 mL of methanol,
Analytical reagent grade of commerce containing not less
previously heated to boiling, and add 300 mL of water at 80°.
than 34.0% w/v and not more than 37.0% w/v of CHzO.
AIIow to cool to room temperature, filter and dry the crystals
A colourIess, aqueous solution with a lachrymatory vapour; at a pressure of 2 kPa.
weight per mL, about 1.08 g.
Store protected from Iight.
Assay Dilute 5 mL to 1000 mL with water. To 10 mL of
the solution add 25 mL of 0.05M iodine VS and 10 mL of
Fuchsin Solution, Basic Dissolve 0.1 g of basic fuchsin in GC Concentrical Column

3 mL of methanol, dilute to 100 mL with water, mix and A commercially available system consisting of 2
filter. concentrically arranged tubes. The outer tube is packed with
Fuchsin Solution, Decolorised Dissolve 0.1 g of basic molecular sieves and the inner tube is packed with a porous
fuchsin in 60 mL of water and add 1 g of anhydrous sodium polyrner mixture. The main application is the separation of
sulfite dissolved in 10 mL of water. Slowly add 2 mL of gases.
hydrochloric acid, stirring continuously, and dilute to 100 mL Gelatin (9000-70-8)
with water. Allow to stand protected from Iight for at least
12 hours. Shake with sufficient activated charco al (0.2 to
0.3 g) to remove the colour and then filter immediately. If Gelatin, Hydrolysed Dissolve 50 g of gelatin in 1000 mL
the solution becomes c1oudy, filter before use. If on standing of water, heat in saturated steam at 121 ° for 90 minutes and
freeze dry.
the solution becomes violet, decolorise again by adding
activated charcoal. Geraniol C lO H 180 = 154.2 (106-24-1)
Complies with the following test. General reagent grade of commerce.
Sensitivity to formaldehyde To 1.0 mL of the solution Boiling point, about 230°; n~o, about 1.476.
add 1.0 mL of water and 0.1 mL of aldehyde-free ethanol Geranyl Acetate (E)- 3, 7-Dimethylocta-2,6-dien-l-yl
(96%). Add 0.2 mL of a solution containing 0.01 % w/v of acetare; C1 2H2002 = 196.3 (105-87-3)
formaldehyde. A pale pink colour is produced within General reagent grade of commerce.
5 minutes.
di~, 0.896 to 0.913; ni?, about 1.463.
Geranyl acetate used in gas chromatography complies with the
Fuchsin Solution Rl, Decolorised To I g of basic fuchsin following test.
add lOO mL of water, hear to 50° and allow to cool, shaking
Assay Examine by gas chromatography as prescribed in
occasionally. A1low to stand for 48 hours, shake and filter.
the monograph for Bitter-orange-f1ower Oi! using the reagent
To 4 mL of rhe filrrate add 6 mL of hydrochloric acid, mix
being examined as the test solution. The area of the
and dilute to 100 mL with water. Allow to stand for at lea sr
principal peak is not les s than 99.0% of the total area of the
1 hour to ensure maximum fading.
peaks.
Fucose See L-Fucose.
Ginsenoside Rbl (20S)-3~-[(2-0-~-D-Glucopyranosyl-~-D
L-Fucose 6-DeoXY-L-galactose; fucos e; glucopyranosyl)oxy]-20-[(6-0-~-D-glucopyranosyl-~-D
C 6 H 12 0 S = 164.2 (6696-41-9) glucopyranosyl)oxyl-5lX-darnmar-24-en-12~-ol;
General reagent grade of commerce. CS4H92023,3H20 = 1163 (41753-43-9)
A whire powder; melting point, abour 140°; [al~, about -76 General reagent grade of commerce.
(9% w/v in water measured after 24 hours). [al~, + 11.3 (in a 1% w/v solution in methanol); melting
FumaricAcid C 4H 4 0 4 = 116.1 (110-17-8) point, about 199°.
General reagent grade of commerce. Water Not more than 6.8%, Appendix IX C, Method 1.
Furfural See Furfuraldehyde. Assay Carry out the liquid chromatographic procedure
Furfuraldehyde Furfural; furan-2-aldehyde; described in the monograph for Ginseng using a 0.03% w/v
C SH 40 2 = 96.09 (98-01-1) solution of the reagent being examined in methanol. The
General reagent grade of commerce. content of ginsenoside Rbl is not less than 95% by
normalisation.
A colourless or pale brownish yellow, oily liquid; boiling
point, about 162°; dig, 1.155 to 1.161. Ginsenoside Re (3~,6lX,12~)-20-(~-D-Glucopyranosyloxy)-
3,12-dihydroxydammar-24-en-6-yl 2-0-(6-deoxy-lX-L-
Galactose See D-Galactose.
mannopyranosyl)-~-D-glucopyranoside;
D-Galactose C 6 H 12 0 6 = 180.2 (59-23-4) C48H 82018 = 947.2 (52286-59-6)
General reagent grade of commerce. General reagent grade of commerce.
A white, crystalline powder; melting point, abour 164°; [al~o, Ginsenoside Rf (20S)-6-0-[~-D-Glucopyranosyl-(I-> 2)-~
abour +80 (10% w/v in water containing about 0.05% of D-glycopyranoside]-darnmar-24-ene-3~, 6lX, 12 ~,20-tetrol;
NH 3)· C42Hn014,2H20 = 837 (52286-58-5)
Gallic Acid 3,4,5-Trihydroxybenzoic acid monohydrate; General reagent grade of commerce.
C 7 H ó O s,H 20 = 188.1 (5995-86-8)
[al~, + 12.8 (in a 1% w/v solution in methanol); melting
General reagent grade of commerce. point, about 198°.
Melting point, about 260°, with decomposition. It loses its Ginsenoside Rgl (20S)-6lX,20-Bis(~-D-glucopyranosyloxy)-
water of crystallisation at 120°. 5lX-darnmar-24-ene- 3~, 12 ~-diol;
Chromatography When examined by test C for C42H n014,2H 20 = 837 (22427-39-0)
Identificarion in the monograph for Bearberry Leaf the General reagent grade of commerce.
chromatogram shows only one spot.
[a]~, + 31.2 (in a 1% w/v solution in methanol); melting
Gallium (68 Ga) Chloride Solution 68GaCI3 = 174.3
point, about 188° to 191 0 .
Solution containing gallium-68 in the form of gallium
chloride in dilute hydrochloric acid.
Water Not more than 4.8%, Appendix IX C, Method 1.
Content, 90% to 11 0% of the declared gallium-68 Assay Carry out the liquid chromatographic procedure
radioacrivity at the date and time stated on the labe!' described in the monograph for Ginseng using a 0.03% w/v
solution of the reagent being examined in methanol. The
Gastric Juice, Artificial Dissolve 2.0 g of sodium chloride
content of ginsenoside Rgl is not les s than 95% by
and 3.2 g of pepsin powder in water. Add 80 mL of
normalisation.
1M hydrochloric acid and dilute to 1000 mL with water.
Ginsenoside Rg2 3~,12~,20-Trihydroxydammar-24-en-6cx L-Glutathione, Oxidised Bis (L-y-glutamyl-

yl 2-0-(6-deoxy-cx-L-mannopyranosyl)-~-D-glucopyranoside; L-cysteinylglycine) disulfide;
C 42 H 72 0 13 = 785 (52286-74-5) C ZOH 3ZN601 ZS2 = 612.6 (27025-41-8)
Gitoxin C41H64014 = 781.0 (4562-36-1) General reagent grade of commerce.
General reagent grade of commerce. Glycerol Propane-l,2,3-triol; C 3H s0 3 = 92.10 (56-81-5)
A white, crystalline powder; melting point, about 283°, with Analytical reagent grade of commerce.
decomposition; [al~o, + 20 to + 24 (0.5% w/v in a mixture of A colourless viscous liquid; weight per mL, about 1.26 g.
equal volurnes of chloroform and methanol) . Glycerol Rl
Complies with the following test. Glycerol complying with the monograph Glycerol and free
Homogeneity Carry out test A for Identification described from diethylene glycol when examined as described in the
in the monograph for Digitalis Leaf applying to the plate a test for Impurity A and related substances in that
solution containing only the reagent being examined. The monograph.
chromatogram shows only one principal spot. Glycerol (85%) Glycerol containing 12.0 to 16.0% w/w of
Glucosamine HydrochIoride water; weight per mL, 1.22 to 1.24 g.
C 6H1 4CINO s = 215 .6 (66-84-2) Glycerol (85 per cent) Rl
Glycerol complying with the monograph Glycerol 85 per cent
[al~o, +100, decreasing to +47.5° after 30 minutes, and free from diethylene glycol when examined as described
determined on a 10% w/v solution in water. in the test for Impurity A and related substances in that
Glucose See D-Glucose. monograph.
o-Glucose Dextrose; C 6H 120 6 = 180.2 (50-99-7) Glycerol l-decanoate (2RS)-2,3-Dihydroxypropyl
Analytical reagent grade of commerce. decanoate; o:-Monocaprin; l-Monodecanoyl-rac-glycerol;
C 13H 26 0 4 = 246.3 (2277-23-8)
A white, crystalline or granular powder; [al~, about +52.5
(10% w/v in water containing about 0.2% v/v of arnmonia). Content, about 99%.
o-Glucose Monohydrate Glycerol l-octanoate (2RS)-2,3-Dihydroxypropyl
C6H1206,H20 = 198.2 (5996-10-1) octanoate; o:-Monocaprylin; l-Monooctanoyl-rac-
General reagent grade of commerce. glycerol;C"H zz 0 4 = 218.3 (502-54-5)
Content, about 99%.
Colourless crystals or a white to cream, crystalline powder;
[al~, about + 52.5 (anhydrous) (10% w/v in water containing GlycidoI C 3H 60 Z = 74.1 (556-52-5)
about 0.2% ofNH 3). General reagent grade of commerce.
o-Glucuronic Acid C 6H 1007 = 194.1 (6556-12-3) d¡O, about 1.115; n~o, about 1.432.
General reagent grade of commerce containing not less than Glycine Aminoacetic acid; C 2H sN0 2 = 75 . 1 (56-40-6)
96.0% of C 6H 10 0 7 , ca1culated with reference to the Analytical reagent grade of commerce.
substance dried in vacuo, Appendix IX D.
Melting point, about 233°, with decomposition.
Shows mutarotation: [al~4, + 11.7 ..... + 36.3. Glycollic Acid 2-Hydroxyacetic acid;
Assay Dissolve 0.15 g in anhydrous methanol while stirring C 2H 40 3 = 76.05 (79-14-1)
and titrate with 0.1 M tetrabutylammonium hydroxide VS, General reagent grade of commerce.
protecting the solution from atmospheric carbon dioxide
throughout the solubilisation and titration. Determine the
Glycyrrhetic Acid See Glycyn'hetinic acid.
end point potentiometrically. Each mL of
O.IM tetrabutylammonium hydroxide VS is equivalent to Glycyrrhetinic Acid Glycyrrhetic acid; a mixture of 0:-
19.41 mg of C 6H 10 0 7 . and ~-isomers with the ~-isomer predominating;
C30H4604 = 470.7 (471-53-4)
Glutamic Acid L-Glutamic acid; C SH 9 0 4 = 147.1 (56-86-
O)
A white to brownish yellow powder; melting point, about
292°, with decomposition; [al~o, +145° to 155° (1 % w/v in
Melting point, about 205°; [al~o, about +31.5 . absolute ethanol) .
Glutarnyl endopeptidase for peptide rnapping Complies with the following requirement.
(137010-42-5) Homogeneity Carry out the method for thin-Iayer
Endoproteinase Glu-C of high purity from Staphylococcus chromatography, Appendix II! A, using a suspension of silica
aureus strain V8 (EC 3.4.21.19). gel F254, prepared using a 0.25% v/v solution of
L-y-Glutarnyl-L-cysteine orthophosphoric acid, to coat the plate and a mixture of
CSH14NzOsS = 250.3 (636-58-8) 5 volumes of methanol and 95 volurnes of chlorofonn as the
General reagent grade of commerce. mobile phase but allowing the solvent front to ascend 10 cm
aboye the line of application. Apply to the plate 5 ¡.¡L of a
Glutaraldehyde CsHsO z = 100.1 (111-30-8)
0.5% w/v solution of the reagent being examined in the
General reagent grade of commerce. mobile phase. After removal of the plate, examine under
Boiling point, about 188°; nf5' about 1.434. ultraviolet light (254 nm). The chromatogram obtained shows
Glutaric Acid Pentanedioic acid; a dark spot with an Rf value of about 0.3 (~
C SH S0 4 = 132.1 (110-94-1) glycyrrhetinicacid) and a smaller spot with an Rf value of
White, crystalline powder. about 0.5(0:-glycyrrhetinic acid). Spray with anisaldehyde
solution andheat at 100 0 to 105' for 10 minutes. Both spots
are bluish violet and between them a smaller bluish violet
spot may be presento
18o:-Glycyrrhetinic Acid (20~)-3~-Hydroxy-11-oxo-18o:- Haemoglobin (9008-02-0)

0Iean-12-en-29-oic acid; C30H4604 = 470.7 (1449-05-4) Content ofnitrogen 15 to 16%.
General reagent grade of commerce. Content ofiron 0.2 to 0.3% .
~-Glycyrrhetinic Acid 3 ~-H ydroxy-ll-oxo-18 ~,20~- Loss on drying Not more than 2% .
0Iean-12-enoic acid; C30H4604 = 470.7 (471-53-4) Sulfated ash Not more than 1.5%.
General reagent grade of commerce. Haemoglobin Solution Transfer 2 g of haemoglobin 10 a
Melting point, about 293°; [O' ]~o, about +170 (1 % w/v in 250 mL beaker and add 75 mL of dilute hydrochlonc acid R2.
chloroform). Stir until solution is complete. Adjust the pH to 1.6 ± 0.1
GlyoxaI Bis(2-hydroxyanil) using 1 M hydrochlonc acid. Transfer 10 a 100 mL ftask with
Bis(2-hydroxyphenylimino) ethane; glyoxalhydroxyanil; the aid of dilute hydrochlonc acid R2. Add 25 mg of
Cl4Hl2N202 = 240.3 thiomersal. Prepare daily, store at 5 ± 3° and readjust to
General reagent grade of commerce. pH 1.6 before use.
Melting point, about 200 0 • Store at 2° to 8 0 •
GlyoxaI Sodium Bisulfite Glyoxal sodium bisulphite; Harpagoside C24H30011 = 494.5
C2H4Na20SS21H20 = 284.2 White or almost white, crystalline powder, very hygroscopic,
General reagent grade of commerce. soluble in water and in ethanol (96%) .
A white or cream powder. Melting point, 117° to 121 0 .
GlyoxaI Solution (107-22-2) Store in an airtight container.
General reagent grade of commerce containing about 40% Hederacoside C 0-6-Deoxy-ex-L-mannopyranosyl-(1-+4)-
w/w of glyoxal (C 2H 2 0 2). O-~-D-glucopyranosyl-( 1-+6)-~-D-glucopyranosyl (4R)- 3~
Assay In a ground-glass-stoppered ftask place 1 g of the [[2-0(-6-deoxy-ex-L-mannopyranosyl)-ex-L-
reagent being examined, 20 mL of a 7% w/v solution of arabinopyranosyl] oxy]-23-hydroxyolean-12-en-28-oate;
hydroxylamine hydrochloride and 50 mL of water. Allow 10 CS9H96026 = 1221 (14216-03-6)
stand for 30 minutes and add 1 mL of methyl red mixed Colourless crystals or white or almost white powder; melting
solution and titrate with 1M sodium hydroxide VS until the point, about 220°.
colour changes from red to green. Carry out a blank titration. Hederacoside e used in liquid chromatography complies with the
Each mL of 1M sodium hydroxide VS is equivalent to following test.
29.02 mg of C 2 H 20 2. Assay Examine by liquid chromatography (2.2.29) as
GlyoxaIbydroxyanil See Glyoxal bis(2-hydroxyanil). prescribed in the monograph on Ivy leaf.
Gonadotrophin, Chorionic (9002-61-3) Test solution Dissolve 5.0 mg of hederacoside e in 5.0 mL
General reagent grade of commerce. of methanol.
A white or almost white, amorphous powder. Content Minimum 95%, calculated by the normalisation
Gonadotrophin, Serum (9002-70-4) procedure.
General reagent grade of commerce. Hederagenin Astrantiagenin E, caulosapogenin,
3 ~,23-dihydroxy-4ex-olean-12-en-28-oic acid;
A white or pale grey, amorphous powder. C30H4S04 = 472.7 (465-99-6)
Guaiacol 2-Methoxyphenol. l-Hydroxy-2- ex-Hederin (+ )-( 4R)-3~-[[2-0-(6-Deoxy-ex-L
methoxybenzene; C 7 H s0 2 = 124.1 (90-05-1) mannopyranosyl) -O:-L-arabinopyranosyl] oxy]-23-
Crystalline mass or colourless or yellowish Iiquid, hydroxyolean-12-en-28-oic acid;
hygroscopic, slightly soluble in water, very soluble in C41H66012 = 751.0 (27013-91-8)
methylene chloride, freely soluble in alcohol. White or almost white powder.
Boiling point, about 205°; melting point, about 28°. Melting point, about 256°.
Guaiacum Resin Gum guaiac Helium See Helium for chromatography
Resin obtained from the heartwood of Guaiacum officinale L. Helium for Chromatography He = 4.003 (7440-59-7)
and Guaiacum sanctum L. Content, minimum 99.995% v/v ofHe.
Reddish brown or greenish brown, hard, glassy fragments; Heparin (9041-08-1)
fracture, shiny. Heparin Sodium of the British Pharmacopoeia.
Guaiazulene 1,4-Dimethyl-7-isopropylazulene; Heptachlor ClOHsCh = 373.3 (76-44-8)
C1sH 1S = 198.3 (489-84-9) Boiling point, about 135°; melting point, about 95° .
General reagent grade of commerce. A suitable certified reference solution (IO ng/).lL in
Dark blue crystals or a blue Iiquid; melting point, about 30°. cyclohexane) may be used.
Store protected from light and air. Heptachlor Epoxide C lO H sCl7 0 = 389.3 (1024-57-3)
Guaiphenesin Guaifenesin; guaiacol glyceryl ether; Boiling point, about 200°; melting point, about 160°.
C lO H l4 0 4 = 198.2 (93-14-1) A suitable certified reference solution (10 ng/).lL in
General reagent grade of commerce. cyclohexane) may be used.
Melting point, about 79°. HeptafIuorobutyric Acid HFBA;
Guanidine Hydrochloride CH sN 3,HCI = 95.5 (50-01-1) C 4 HF7 0 2 = 214.0 (375-22-4)
General reagent grade of commerce. Clear, colourless Iiquid. Corrosive.
Guanine 2-Aminopurin-6-one; CsHsNsO = 151.1 (73-40- d~g, about 1.645; n~, about 1.300; boiling point, about
5) 120°.
General reagent grade of commerce. Content, minimum 99.5% .
Heptafluorobutyric Anhydride C SF I4 0 3 = 410.1 (336- 1,1,1,3,3,3-Hexafluoropropan-2-o1

59-4) C3HzF60 = 168.0 (920-66-1)
Use a grade of commerce suitable for derivatisation. Content, minimum 99.0%, determined by gas
Boiling point, about 108°; nfjl, about 1.287. chromatography.
Heptafluoro-N-methyI-N-(trimethyIsilyI)butanamide Clear, colourless liquid, miscible with water and with
2,2,3,3,4,4,4-Heptafluoro-N-methyl-N- anhydrous ethano!.
(trimethylsilyl) buryramide; dig, about 1.596; boiling point, about 59".
C sH 1zF 7NOSi = 299.3 (53296-64-3) Hexamethyldisilazane C6Hl 9NSiz = 161.4 (999-97-3)
Clear, colourless liquid, flammable. Clear, colourless liquido
nfjl, about 1.351; boiling point, about 148°. General reagent grade of commerce.
Heptane C 7HI6 = 100.2 (142-82-5) dig, about 0.78; nbo, about 1.408; boiling point, about 125°.
Colourless, flammable liquid, practically insoluble in water, Store in an airtight container.
miscible with anhydrous ethano!. Hexamethylenetetramine Hexamine, 1,3,5,7-tetra-
dig, 0.683 to 0.686; nbo, about 1.387 to 1.388. azatricyc10 [3.3.1.1 3 ,7]decane; C 6H 12N 4 = 140.2 (100-97-0)
Distillation range (2.2.11). Not les s than 95 % distils Colourless, crystalline powder, very soluble in water.
between 97° and 98°. Hexamine See Hexamethylenetetramine
n-Heptane See Heptane Hexane C 6H I4 = 86.2 (110-54-3)
2-Heptylamine 2-Aminoheptane; C 7H17N = 115.2 Colourless, flammable liquid, practically insoluble in water,
General reagent grade of commerce. miscible with anhydrous ethano!.
Boiling point, about 143°. dig, 0.659 to 0.663; nfjl, 1.375 to 1.376.
Hesperidin (S)-7-[[(6-0-(6-Deoxy-ex-L-mannopyranosyl)- Distillation range (2.2.11). Not less than 95% distils
~-D-glucopyranosyl] oxy] -5-hydroxy-2-(3-hydroxy-4- between 67° and 69°.
methoxyphenyl) -2,3-dihydro-4H-1-benzopyran-4-one; Hexane used in spectrophotometry complies with the f ollowing
CZSH3401 S = 611 (520-26-3) additional test.
Hygroscopic powder, slightly soluble in water and in Minimum transmittance (2.2.25) using water as
methano!. compensation liquid: 97% from 260 nm to 420 nm .
Melting point, 258° to 262°. n-Hexane C 6H l4 = 86.2 (110-54-3)
Hexachlorobenzene C 6CI 6 = 284.8 (118-74-1) Analytical reagent grade of commerce usually containing not
Boiling point, about 332°; melting point, about 230°. less than 99 % of the pure isomer.
A suitable certified reference solution (1 O ngl~IL in A colourless, flammab1e liquid; boiling point, about 68°;
cyc1ohexane) may be used. weight per mL, about 0.66 g.
ex-Hexachlorocyc1ohexane C 6H 6 CI 6 = 290.8 (319-84-6) Hexane, Purified A grade of hexane containing not more
Boiling point, about 288°; melting point, about 158°. than 0.002% w/v of non-volatile matter.
A suitable certified reference solution (10 ngl¡.tL in Hexylamine C 6H 1S N = 101.2 (111-26-2)
cyc1ohexane) may be used. Colourless liquid, slightly soluble in water, soluble in ethanol
~-Hexachlorocyc1ohexane C 6H 6CI 6 = 290 .8 (319-85-7) (96%).
A suitable certified reference solution (10 ngl¡.tL in dig, about 0.766; nbo, 1.418; boiling point, 127° to 131 °.
cyc1ohexane) may be used. Histamine Dihydrochloride
8-Hexachlorocyc1ohexane C 6H 6CI 6 = 290.8 (319-86-8) C SH 9N 3 ,2HCI = 184.1 (56-92-8)
A suitable certified reference solution (10 ngl¡.tL in Of the British Pharmacopoeia.
cyc1ohexane) may be used. Histamine Phosphate Histamine acid phosphate;
Hexacosane C z6H s4 = 366.7 (630-01-3) CSH9N3,2H3P04 = 307.1 (23297-93-0)
Colourless or white or almost white flakes. Of the British Pharmacopoeia.
Melting point, about 5r . Histamine SoIution A 0.9 % w/v solution of sodiwn
Hexadimethrine Bromide 1,5-Dimethyl-1,5- chloride containing 0.1 ¡.tg per mL of histamine base, C SH 9N 3
diazundecamethylene polymethobromide; poly( 1,1,5,5- (as the phosphate or the dihydrochloride).
tetramethyl-1,5-azoniaundecamethylene dibromide); Histidine Monohydrochloride (RS)-2-Amino-3-
(C I3H30BrzNz)" (28728-55-4) (imidazol-4-yl)propionic acid hydrochloride monohydrate;
White or almost white, amorphous powder, hygroscopic, C 6H g N 3 0z,HCl,H zO = 209.6 (123333-71-1)
soluble in water. Crystalline powder or colourless crystals, soluble in water.
Store in an airtight container. Melting point, about 250°, with decomposition.
2,2 ' ,2 " ,6,6' ,6"-Hexa-(1,1-dimethylethyI)-4,4' ,4 " -[2,4,6- Chromatography Thin-layer chromatography (2.2.27) as
trimethyI-l,3 ,5-benzenetriyltrismethy1ene ] -triphenoI prescribed in themonograph Histamú¡e dihydrochloride (0143);
2,2' ,2" ,6,6 ' ,6"-Hexa-tert-buryl-4,4' ,4"- [(2,4,6-trimethyl- the chromatogram shows only one principal spot.
1,3,5-benzenetriyl)trismethylene]triphenol; C s4H 78 0 3 = 775 Holmium Oxide Diholmium trioxide;
Crystalline powder, practically insoluble in water, soluble in HOZ0 3 = 377.9 (12055-62-8)
acetone, slightly soluble in ethanol (96%) . Yellowish powder, practically insoluble in water.
Melting point, about 244 0
•
Holmium Perchlorate SoIution
A 4% w/v solution of holmium oxide in a solution of perchloric
acid containing 14.1% w/v ofHCI0 4 .
2014 Appendix 1 A V -A 71
DL-Homocysteine (2RS)-2-Amino-4-sulfanylbutanoic acid; colourless, constant-boiling mixture (55% 10 58% of HI)

C 4 H 9 N 0 2S = 135.2 (454-29-5) 0
distilling between 126 and 127" .
White or almost white, crystalline powder. Place the acid in small, amber, glass-s1Oppered bottles
Melúng point, about 232°. previously flushed with caJ'bon dioxide or nitrogen, seal with
L-Homocysteine Thiolactone Hydrochloride (3S)-3- paraffin.
Aminodihydrothiophen-2(3H)-one hydrochloride; Store in a dark place.
C 4 H sClNOS = 153.6 (31828-68-9) Hydrobromic Acid, 30 per cent
White or almost white, crystalline powder. HBr = 80.92 (10035-10-6)
Melting point, about 202°. A 30% solution of hydrobromic acid in glacial acetic acid R.
Honokiol 3' ,5-Di(prop-2-enyl)biphenyl-2,4'-diol, 3 ',5- Degas with caution the contents before opening.
Diallyl-2,4'-dihydroxybiphenyl, 3 ',5-Di-2-propenyl-[I,I '- Hydrobromic Acid, 47 per cent
biphenyl]-2,4 '-diol; ClsH1S02 = 266.3 (35354-74-6) HBr = 80.92 (10035-10-6)
Hyaluronate Solution Dilute potassium hyaluronate stock A 47% m/m solution of hydrobromic acid.
soluáon with an equal volume of phosphate-buffered satine Hydrobromic Acid, Dilute Place 5.0 mL of 30%
pH 6.4. hydrobromic acid in amber vials equipped with polyethylene
U se on the day of preparaúon. s1Oppers. Seal under argon and s10re in the dark.
Hyaluronidase Diluent Add 5.0 mL of glacial acetic acid immediately before use.
Mix 100 mL of phosphate buffer solution pH 6.4 with 100 mL Shake.
of water. Dissolve 0.140 g of hydrolysed gelatin in the solution Store in the dark.
at 37°. Hydrobromic Acid Rl, Dilute
Use within 2 h. Contains 0.79% w/v of HBr.
Hydrastine Hydrochloride (3S)-6, 7-Dimethoxy-3-[ (5 R)- Dissolve 16.81 g of 47% hydrobromic acid in water and dilute
6-methyl-5,6, 7,8-tetrahydro-l ,3-dioxolo [4 ,5-g] isoquinolin-5- 10 1000 mL with the same solvento
yl] isobenzofuran-l (3H)-one hydrochloride; C 21 H 22 ClN0 6 = Hydrocarbons (Type L), Low-vapour-pressure
419.9 (5936-28-7)
Unctuous mass, soluble in benzene and in toluene.
White or almost white powder, hygroscopic, very soluble in
Hydrochloric Acid HCl = 36.46 (7647-01-0)
water and in ethanol (96%).
Where no molarity is indicated use analytical reagent grade of
[a]g, about + 127; melting point, about 116°.
commerce with a relative density of about 1.18, containing
Hydrastine hydrochloride used in liquid chromatography complíes not less than 35% w/w and not more than 38% w/w of HCl
with the following additional test. and about 11.5M in strength.
Assay Liquid chromatography (2.2.29) as prescribed in the A colourless, fuming liquido
monograph Goldenseal rhizome (1831).
Solutions of molarity XM should be prepared by diluting 85x
Content, minimum 98%, ca1culated by the normalisation mL of hydrochlon'c acid 10 1000 mL with water.
procedure.
S10re in a container of polyethylene or other non-reacting
Hydrazine Diazine; H 4 N 2 = 32.05 (302-01-2) material at a temperature not exceeding 30°.
Slightly oily liquid, colourless, with a strong odour of Hydrochloric Acid, Brominated To 1 mL of bromine
ammonia, miscible with water. Dilute solutions in water are solution add 100 mL of hydrochloric acid.
commercially available.
Hydrochloric Acid, Dilute
nj3l, about 1.470; boiling point, about 113°; melúng point, Contains 7.3% w/v ofHCI.
about 1.5°.
Dilute 20 g of hydrochloric acid to 100 mL with water.
Caution toxic and corrosive.
Hydrochloric Acid, Dilute, Heavy Metal-free Complies
Hydrazine Hydrate N 2H 4 ,H20 = 50.06 (10217-52-4) with the requirements prescribed for dilute hydrochloric acid
Analyúcal reagent grade of commerce. with the following maximum contents of heavy metals.
A colourless liquidó weight per mL, about 1.03 g. As: 0.005 ppm; Cd: 0.003 ppm; Cu: 0.003 ppm; Fe:
Hydrazine Sulfate Hydrazine sulphate; 0.05 ppm; Hg: 0.005 ppm; Ni: 0.004 ppm; Pb: 0.001 ppm;
N zH 4 ,H 2S0 4 = 130.1 (10034-93-2) Zn: 0.005 ppm.
Colourless crystals, sparingly soluble in cold water, soluble in Hydrochloric Acid, Ethanolic Solutions of the requisite
hot water (50°) and freely soluble in boiling water, practically molaritymay be obtained by diluting hydrochloric acid with
insoluble in ethanol (96%). ethanol (96%) in place of water as directed under hydrochloric
Arsenic (2.4.2, Method A): maximum 1 ppm, determined acid. If no molarity is stated, use a solution prepared by
on 1.0 g. diluting 5 mL of 1M hydrochloric acid to 500 mL with ethanol
(96%).
Sulfated ash (2.4. 14) : maximum 0.1 %.
Hydrindantin 2,2 ' -Dihydroxy-2,2 '-bi-indan-l, l ' ,3,3'- Hydrochloric Acid, Heavy Metal-free Complies with
tetraone dihydrate; ClsHIO06,2H20 = 358.3 (5950-69-6) the requirements prescribed for hydrochloric acid with the
following maximum contents of heavy metals.
As : 0.005 ppm; Cd: 0.003 ppm; Cu: 0.003 ppm; Fe:
Melting point, about 258°. 0.05 ppm; Hg: 0.005 ppm; Ni: 0.004 ppm; Pb: 0.001 ppm;
Hydriodic Acid HI = 127.9 (10034-85-2) Zn: 0.005 ppm.
Prepare by distilling hydriodic acid over red phosphorus, Hydrochloric Acid, Lead-free
passing carbon dioxide or nitrogen through the apparatus Complies with the requirements prescribed for hydrochloric
during the distillation. Use the colourless or almost acid with the following additional requirement.
Lead: maximum 20 ppb. Hydrogen Peroxide Solution (20 vol)

Atomic emission spectrometry (2.2.22, Method l) . Analytical reagent grade of commerce containing about
Test solution. In a quartz crucible evaporate 200 g of the acid 6% w/v of H 2 0 2 or hydrogen peroxide solution (100 vol)
to be examined almost to dryness. Take up the residue in diluted with 4 volumes of water.
5 mL of nitric acid prepared by sub-boiling distillation of A colourless liquid; weight per mL, about 1.02 g.
nitric acid and evaporate to dryness. Take up the residue in Hydrogen Peroxide Solution (10 vol) See Dilute
5 mL of nitric acid prepared by sub-boiling distillation of hydrogen peroxide solution
nitric acid. Hydrogen Peroxide Solution, Dilute Hydrogen Peroxide
Reference solutions. Prepare the reference solutions using lead Solution (3 per cent) of the British Pharmacopoeia.
standard solution (0.1 ppm Pb) diluted with nitric acid Hydrogen Peroxide Solution, Strong Hydrogen
prepared by sub-boiling distillation of nitric acid. Peroxide Solution (30 per cent) of the British
Wavelength: 220.35 run. Pharmacopoeia.
Hydrochloric Acid, Methanolic Solutions of the Hydrogen Sulfide Hydrogen sulphide;
requisite molarity may be obtained by diluting hydrochloric H 2S = 34.08 (7783-06-4)
acid with methanol in place of water as directed under Gas, slightly soluble in water.
hydrochloric acid.
Caution: poisonous gas.
Hydrochloric Acid R1
Hydrogen Sulfide Rl Hydrogen sulphide Rl
Contains 25% w/v of HC!.
Content, minimum 99.7% v/v.
Dilute 70 g of hydrochloric acid to 100 mL with water.
Hydrogen Sulfide Solution Hydrogen sulphide solution
Hydrochloric Acid R1, Dilute
A recently prepared solution of hydrogen sulfide in water.
Contains 0.037% w/v of HC!. The saturated solution contains about 0.4% to 0.5% of H 2 S
Dilute 1.0 mL of dilute hydrochloric acid to 200.0 mL with at 20°.
water. Hydroquinone Quinol; benzene-l,4-diol;
Hydrochloric Acid R2, Dilute Dilute 30 mL of C6H60Z = 110.1 (123-31-9)
1M hydrochloric acid to 1000 mL with water; adjust to Fine, colourless or white or almost white needles, darkening
pH 1.5 to 1.7 . on exposure to air and light, soluble in water and in ethanol
Hydrochloric Acid, Stannated (96%).
Use sta=ated hydrochloric acid low in arsenic, grade of Melting point, about 173°.
commerce, or prepare by adding 1 mL of tin(lI) chloride Store protected from light and air.
solution to 100 mL of hydrochloric acid.
Hydroquinone Solution
Hydrocortisone CZ¡H300S = 362.5 (50-23-7)
Dissolve 0.5 g of hydroquinone in water, add 20 IlL of sulfuric
General reagent grade of commerce. acid and dilute to 50 mL with water.
Melting point, about 214°, with decomposition. 4-Hydroxybenzaldehyde C7 H 60 2 = 122.2 (123-08-0)
Hydrocortisone Acetate C23H 3206 = 404.5 (50-03-3) General reagent grade of commerce.
Of the British Pharmacopoeia. Colourless needles; melting point, about 118°.
Hydroftuoric Acid HF = 20.01 (7664-39-3) 2-Hydroxybenzimidazole lH-benzirnidazol-2-ol;
Content, minimum 40.0% mlm. C 7 H 6N 2 0 = 134.1 (615-16-7)
Clear, colourless liquido 4-Hydroxybenzohydrazide p-Hydroxybenzohydrazide;
Loss on ignition: not more than 0.05% mlm; evaporate C 7 H sN 20 2 = 152.2 (5351-23-5)
the hydrofluoric acid in a platinum crucible and gently ignite 4-Hydroxybenzoic Acid Parahydroxybenzoic acid;
the residue to constant mass. C 7 H 60 3 = 138.1 (99-96-7)
Assay Weigh accurately a glass-stoppered flask containing Crystals, slightly soluble in water, very soluble in ethanol
50.0 mL of 1 M sodium hydroxide. Introduce 2 g of the (96%), soluble in acetone.
hydrofluoric acid and weigh again. Titrate the solution with Melting point, 214° to 215°.
0.5 M sulfun'c acid, using O.5mL of phenolphthalein solution as
indicator. 4-Hydroxybiphenyl See Biphenyl-4-o1
1 mL of 1 M sodium hydroxide is equivalent to 20.01 mg of 4-Hydroxycoumarin 4-Hydroxy-2H-l-benzopyran-2-one;
HF. C 9 H 60 3 = 162.1 (1076-38-6)
Store in a polyethylene container. White or almost white powder, freely soluble in methano!.
Hydrogen See Hydrogen for chromatography Content, minimum 98.0%.
Hydrogen for Chromatography H 2 = 2.016 (1333-74-0) 6-Hydroxydopa (2RS)-2-Amino-3-(2,4,5-
trihydroxyphenyl)propanoic acid; 2,5-DihydroxY-DL-tyrosine;
Content, minimum 99.95% v/v. C 9 H¡¡NO s = 213 .2 (21373-30-8)
Hydrogen Peroxide Solution (200 vol) 0
H 20 2 = 34.02 (7724-84-1)
2-[4- (2-Hydroxyethyl)piperazin-l-yl]ethanesulfonic
General reagent grade of commerce containing about Acid HEPES, 2-[4-(2-hydroxyethyl)piperazin-l-
60% w/v of H Z0 2' y~ethanesulphonic acid; C sH¡ sN 2S0 4 = 238.3 (7365-45-9)
A colourless liquid; weight per mL, about 1.18 g. White or almost white powder.
Hydrogen Peroxide Solution (100 vol) See Strong Melting point, about 236°, with decomposition.
hydrogen peroxide solution
4-Hydroxyisophthalic Acid 4-Hydroxybenzene-1,3- White or almost white powder.

dicarboxylic acid; CSH 60 S = 182.1 (636-46-4) Melting point, 71 ° to 74°.
Needles or platelets, very slightly soluble in water, freely 5-Hydroxyuracil Isobarbituric acid; pyrimidine-2,4,5-triol;
soluble in ethanol (96%). 2,4,5-trihydroxypyrimidine; C 4 H 4 N 20 3 = 128.1 (496-76-4)
Melting point, about 314°, with decomposition. White or almost white, crystalline powder.
Hydroxy1amine Hydrochloride Hydroxylammonium Melting point, about 310°, with decomposition.
chloride; NH 20 H,HCl = 69.49 (5470-11-1)
Hornogeneity Carry out the method described under
White or almost white, crystalline powder, very soluble in Related substances in the monograph for Fluorouracil. The
water, soluble in ethanol (96%). chromatogram exhibits a principal spot with an Rf value of
Hydroxylamine Hydrochloride Solution R2 Dissolve about 0.3.
2.5 g of hydroxylamine hydrochloride in 4.5 mL of hot water Store in an airtight container.
and add 40 mL of ethanol (96%) and 0.4 mL of bromophenol
Hyoscine Hydrobromide Scopolamine hydrobromide;
blue solution R2. Add O.5M ethanolic potassium hydroxide until a C 17 H 2¡N04 ,HBr,3H 20 = 438.3 (114-49-8)
greenish yellow colour is obtained. Dilute to 50 mL with
ethanol (96%). Of the British Pharmacopoeia.
Hydroxylamine Solution, Alcoholic Dissolve 3.5 g of Hyoscyamine Sulfate Hyoscyamine sulphate;
hydroxylamine hydrochloride in 95 mL of ethanol (60%), add (CI7H23N03h,H2S04,2H20 =: 712 .8 (620-61-1)
0.5 mL of a 0.2% w/v solution of methyl orange in ethanol Of the British Pharmacopoeia.
(60%) and sufficient O.5M potassium hydroxide in ethanol Hypericin 1,3,4,6,8, 13-Hexahydroxy-l0,11-
(60%) to give apure yellow colour. Dilute to 100 mL with dimethylphenanthro [1,10, 9,8-opqra]perylene-7, 14-dione;
ethanol (60%). C30H¡60S = 504.4 (548-04-9)
Hydroxylamine Solution, Alkaline Immediately before Content, minimum 85%.
use, mix equal volumes of a 13.9% w/v solution of Hyperoside 2-(3,4-Dihydroxyphenyl)- 3-~-D
hydroxylamine hydrochloride and a 15% w/v solution of sodium galactopyranosy loxy-5, 7-dihydroxy-chromen-4-one;
hydroxide. C 2I H 200¡2 =: 464.4
Hydroxylamine Solution Rl, Alkaline Faint yellow needles, soluble in methanol.
Solution A Dissolve 12.5 g of hydroxylamine hydrochloride Melting point, about 240°, with decomposition.
in methanol and dilute to 100 mL with the same solvento
Absorbance (2.2.25). A solution in methanol shows two
Solution B Dissolve 12.5 g of sodium hydroxide in methanol absorption maxima at 259 nm and at 364 nm.
and dilute to 100 mL with the same solvento
Hypophosphorous Reagent Dissolve, with the aid of
Mix equal volumes of solution A and solution B irnmediately gentle heat, 10 g of sodium hypophosphite in 20 mL of water
before use. and dilute to 100 mL with hydrochloric acid. Allow to settle
Hydroxymethylfurfural 5-Hydroxyrnethylfurfuraldehyde; and decant or filter through glass wool.
C 6H 60 3 = 126.1 (67-47-0) Hypoxanthine 6-Hydroxypurine, 1H-Purin-6-one;
Acicular crystals, freely soluble in water, in acetone and in C SH 4 N 4 0 =: 136.1 (68-94-0)
ethanol (96%). White or almost white, crystalline powder, very slightly
Melting point, about 32°. soluble in water, sparingly soluble in boiling water, soluble in
Hydroxynaphthol Blue, Sodium Salt Trisodium dilute acids and in dilute alkali hydroxide solutions,
2,2/-dihydroxy-1, l/-azonaphthalene-3' ,4,6/-trisulfonate; decomposes without melting at about 150°.
C 20 H¡ ¡N2Na30¡ ¡S3 = 620 (63451-35-4) Chrornatography Thin-layer chromatography (2.2.27) as
General reagent grade of commerce. prescribed in the monograph Mercaptopurine (0096); the
4- (4-Hydroxyphenyl) butan-2-one chromatogram shows only one principal spot.
CIOH¡202 = 164.2 (5471-51-2) lmidazole Glyoxaline; C 3H 4 N 2 =: 68.1 (288-32-4)
General reagent grade of commerce. White or almost white, crystalline powder, soluble in water
Melting point, about 82°. and in ethanol (96%).
2-Hydroxypropylbetadex for Chromatography Melting point, about 90°.
Betacyc10dextrin modified by the bonding of (R) or (RS) lmidazole, RecrystalHsed Twice recrystallise 25 g of
propylene oxide groups on the hydroxyl groups. imidazole from 100 mL of toluene, cool in ice with stirring,
Hydroxypropyl-~-cyclodextrin (94035-02-6)
wash with ether and dry at room temperature at a pressure of
2 kPa over anhydrous silica gel, or use a purified grade of
Hydroxypropylbetadex of the British Pharmacopoeia.
commerce.
pH (2.2.3): 5.0 to 7.5 for a 2.0% w/v solution. Complies with the following test.
Hydroxyquinoline 8-Hydroxyquinoline; Quinolin-8-ol;
Light absorption Absorbance of an 8% w/v solution at
C 9 H 7 NO = 145 .2 (148-24-3)
325 nm, not more than 0.10, Appendix II B.
White or slightly yellowish, crystalline powder, slightly
lmidazole Solution Dissolve 8.25 g of recrystallised
soluble in water, freely soluble in acetone, in ethanol (96%)
imidazole in 60 mL of water, adjust the pH to 6.75 to 6.85
and in dilute mineral acids.
with 5M hydrochloric acid and add sufficient water to produce
Melting point, about 75°. 100 mL.
I
Sulfated ash (2.4.14): maximum 0.05%. The hydrochloric acid used in preparing this reagent must be
8-Hydroxyquinoline See Hydroxyquinoline. free from stabilising mercury compounds.
12-Hydroxystearic Acid 12-Hydroxyoctadecanoic acid; lmidazole-Mercury Reagent Dissolve 8.25 g of
C¡ SH 36 0 3 = 300.5 (106-14-9) recrystallised imidazole in 60 mL of water and add 10 mL of
5M hydrochloric acid. Stir the solution magnetically and add, lodine-123 and Rutheniurn-l06 Spiking Solution
dropwise, 10 mL of a 0.27% w/v solution of mercury(Il) Prepare immediately before use. Mix 3.5 mL of an 18.5
chloride. If a c10udy solution results, discard and prepare a kBq/mL solution of ruthenium-l 06 in the form of ruthenium
further solution adding the mercury chloride solution more trichloride in a mixture of equal volumes of glacial acetic acid
slowly. Adjust the pH to 6.75 to 6.85 with 5M hydrochloric and water with 200 J.lL of a 75 kBq/mL solution of
acid (about 4 mL is required) and add sufficient water to iodine-123 in the form of sodium iodide in water.
produce 100 mL. lodine Bromide IBr = 206.8 (7789-33-5)
lminodibenzyl 10, ll-Dihydrodibenz[bJ]azepine; Bluish-black or brownish-black crystals, freely soluble in
C 14H 13 N = 195.3 (494-19-9) water, in ethanol (96%) and in glacial acetic acid.
Pale yellow, crystalline powder, practically insoluble in water,
Boiling point, about 116°; melting point, about 40°.
freely soluble in acetone .
lodine Bromide Solution Dissolve 20 g of iodine bromide
Imperatorin 9-[ (3-Methylbut-2-enyl)oxy)-7 H-furo [3,2-
in glacial acetic acid and dilute to 1000 mL with the same
g)[1)benzopyran-7-one; C 1óH 14 0 4 = 270.3 (482-44-0)
solvent.
2-lndanamine hydroehloride 2-Aminoindane
lodine Ch10ride ICI = 162.4 (7790-99-0)
hydrochloride; C 9H 1Z CIN = 169.7 (2338-18-3)
Black crystals, soluble in water, in acetic acid and in ethanol
2,3-Dihydro-lH-inden-2-amine hydrochloride. (96%).
Indigo Carmine CI 73015; acid blue 74; Boiling point, about 97.4°.
ClóHsNzNazOsSz = 466.3 (860-22-0)
lodine Ch10ride Solution Dissolve 1.4 g of iodine chloride
It usually contains sodium chloride.
in glacial acetic acid and dilute to 100 mL with the same
Blue or violet-blue powder or blue granules with a coppery acid.
lustre, sparingly soluble in water, practically insoluble in
ethanol (96%) . Ir is precipitated from an aqueous solution by
sodium ch1oride. lodine Monoeh1oride Reagent, Strong Dissolve 10 g of
potassium iodide and 6.4 g of potassium iodate in 75 mL of
Indigo Carmine Solution To a mixture of 10 mL of
water, add 75 mL of hydrochloric acid and 5 mL of chloroform,
hydrochloric acid and 990 mL of 20% w/v nitrogen-free sulfuric shake and, if necessary, add dropwise with vigorous shaking
acid add 0.2 g of indigo carmine.
O.IM potassium iodide until a faint iodine colour appears in the
The solution complies with the follo wing test Add 1O mL to a chloroform layer. Add in the same manner O.OOI M potassium
solution of 1.0 mg of potassium nirrate in 10 mL of water, iodate until the chloroform is just colourless.
rapidly add 20 mL of nitrogen-free sulfuric acid and heat to
Before use, readjust with either O. IM potasSiW17 iodide or
boiling. The blue colour is discharged within l minute.
O.OOIM potassium iodate as required.
Indigo Carrnine Solution Rl Dissolve 4 g of indigo
Store in a cool place protected from Iight.
carmine in about 900 mL of water added in several portions.
Add 2 mL of sulfuric acid and dilute to 1000 mL with water. lodine Monoch1oride Solution Dissolve 8 g of iodine
trichloride in about 200 mL of glacial acetic acid and
Assay Place in a 100 mL conical fiask with a wide neck separately dissolve 9 g of iodine in 300 mL of dichloromethane.
10.0 mL of nitrate standard solution (100 ppm NOJJ, 10mL of
Mix the two solutions and dilute to 1000 mL with glacial
water, 0.05 mL of the indigo carmine solution R1, and then in
acetic acid.
a single addition, but with caution, 30 mL of sulphuric acid.
Titrate the solution irnmediately, using the indigo carmine Store in a stoppered bottle, protected from Iight and at a
solution R1, until a stable blue colour is obtained. temperature not exceeding 15°.
The number of millilitres used, n, is equivalent to 1 mg of lodine Pentoxide, Recrystallised Di-iodine pe~toxide,
iodic anhydride, iodine pentoxide;
N0 3 •
IzOs = 333.8 (12029-98-0)
Indometacin C1 9HlóCIN04 = 357.8 (53-86-1)
Content, minimum 99.5 %.
White or almost white, crystalline powder, or white or
Industrial Methylated Spirit (95%) Of the British
greyish-white granules, hygroscopic, very soluble in water
Pharmacopeia.
forming HI0 3 .
Inosine 9-~-D-Ribofuranosylhypoxanthine; 9-~-D
Stability on heating Dissolve 2 g, previously heated for
Ribofuranosyl-l,9-dihydro-6H-purin-6-one;
1 hour at 200°, in 50 mL of water. A colourless solution is
ClOH1ZN40S = 268.2 (58-63-9)
obtained.
Assay Dissolve 0.190 g in 50 mL of water, add 3 g of
myo-Inositol Of the British Pharmacopeia. potassium iodlde and ¡JO mL of dilute hydrochloric acid. Titrate
lodie Acid HI0 3 = 175.9 (7782-68-5) the Iiberated iodine with 0.1 M sodium thiosulfate, using 1 mL
Analytical reagent grade of commerce. of starch solution as indicator.
lodine Iz = 253.8 (7553-56-2) 1 mL of 0.1 M sodium thiosulfate is equivalent to 2.782 mg of
Of the British Pharmacopoeia. IzOs·
To prepare 0.05M iodine dissolve 20 g of potassium iodide in Store in an airtight container, protected from light.
the minimum amount of water, add 13 g of iodine, allow to lodine Solution, Alcoholic A 1% w/v solution of iodine in
dissolve and add sufficient water to produce 1000 mL. ethanol (96%).
Weaker solutions should be prepared using proportionately Store protected from light.
les ser amounts of reagents or by appropriate dilution.
Iodine Solution, Chloroformic A 0.5% w/v solution of 20 volumes of water,40 volumes of glacial acetic acid and
iodine in chloroform. 40 volumes of toluene.Allow the plate to dry in air and
Store protected from light. examine in ultraviolet light at254 nm. The chromatogram
shows only one principal spot.
Iodine Solution Rl To 10.0 mL of 0.05M iodine add 0.6 g
of potassium iodide and dilute to 100.0 mL with water. Iodomethane See Methyl Iodide
Prepare immediately before use. Iodoplatinate Reagent To 3 mL of a 10% w/v solution of
Iodine Solution R2 To 10.0 mL of 0.05M iodine add 0.6 g chloroplatinic(IV) acid add 97 mL of water and 100 mL of a
of potassium iodide and dilute to 1000.0 mL with water. 6% w/v solution of potassium iodide.
Prepare immediately before use. Store prótected from light.
Iodine Solution R3 Dilute 2.0 mL of iodine solution Rl to Iodoplatinate Reagent Rl
100.0 mL with water. Prepare immediately before use. Mix 2.5 mL of a 50 gIL solution of chloroplatinic acid,
Iodine Solution R4 Dissolve 14 g of iodine in 100 mL of 22.5 mL of a 100 giL solution of potassium iodide and 50 mL
a 40% w/v solution of potassium iodide, add 1 mL of dilute of water.
hydrochloric acid and dilute 10 1000 mL with water. Store protected from light, at a temperature of 2° to 8°.
Store protected from light. 2-Iodopropane See Isopropyllodide
Iodine Trichloride ICI3 = 233 .3 (865-44-1) Iodosulfurous Reagent See Karl Fischer reagent VS.
Analytical reagent grade of commerce. 5-Iodouracil 5-lodo-lH,3H-pyrimidine-2,4-dione;
Reddish orange crystals. C4H31NzOz = 238.0 (696-07-1)
Iodoacetic Acid CzH310z = 185.9 (64-69-7) Melting point, about 276°, with decomposition.
Colourless or white or almost white crystals, soluble in water Chromatography Thin-Iayer chromatography (2.2.27) as
and in ethanol (96%). prescribed in the monograph Idoxuridine (0669): apply 5 ¡.tL
Melting point, 82° 10 83°. of a 0.25 gIL solution; the chromatogram obtained shows
only one principal spot.
2-Iodobenzoic Acid C 7 HsIOz = 248.0 (88-67-5)
Ion-exchange Resin, Strongly Acidic Resin in
White or slightly yellow, crystalline powder, slightly soluble in
protonated form with sulfonic acid groups attached to a
lattice consisting of polystyrene cross-linked with 8 % of
Melting point, about 160°. divinylbenzene. It is available as spherical beads; unless
Chromatography Thin-Iayer chroma1Ography (2.2.27), otherwise prescribed, the particle size is 0.3 mm to 1.2 mm.
using cellulose for chromatography fZS4 as the coating A strongly acidic resin in bead form (0.3 mm to 1.2 mm) .
substance: apply to the plate 20 ¡.tL of a solution of the Capacity 4.5 mmol to 5 mmol per gram, with a water
2-iodobenzoic acid, prepared by dissolving 40 mg in 4 mL of content of 50 to 60%.
0.1 M sodiumhydroxide and diluting to 10 mL with water.
Preparation 01 a column Unless otherwise prescribed,
Develop oyera path of about 12 cm using as the mobile
use a tube with a fused-in sintered glass disc having a length
phase the upperlayer obtained by shaking together
of 400 mm, an internal diameter of 20 mm and a filling
20 volumes of water,40 volumes of glacial acetic acid and
height of about 200 mm. Introduce the resin, mixing it with
40 volumes of toluene.Allow the plate to dry in air and
water and pouring the slurry into the tube, ensuring that no
examine in ultraviolet light at254 nm. The chromatogram
air bubbles are trapped between the particles. When in use,
shows only one principal spot.
the liquid must not be allowed to fall below the surface of
3-Iodobenzylammonium Chloride 1- the resino If the resin is in its protonated form, wash with
(3-lodophenyl)methanarnine hydrochloride; 1- water until 50 mL requires not more than 0.05 mL of 0.1 M
(3-iodophenyl)methanaminium chloride; m-iodobenzylamine sodium hydroxide for neutralisation, using 0.1 mL of methyl
hydrochloride; C 7 H 9 CIIN = 269.5 (3718-88-5) orange solution as indicator.
0
White or almost white crystals; melting point, 188 to 190°. If the resin is in its sodium form or if it requires regeneration,
Iodoethane CzHsI = 155.9 (75-03-6) pass about 100 mL of a mixture of equal volumes of
Colourless or slightly yellowish liquid, darkening on exposure hydrochloric acid R1 and water slowly through the column and
to air and light, miscible with ethanol (96%) and most then wash with water as described aboye.
organic solvents. Ion-exclusion Resin for Chromatography
di8, about 1.95; nj5l, about 1.513; boiling point, about 72°. A res in with sulfonic acid groups attached to a polymer
Store in an airtight container. lattice consisting of polystyrene cross-linked with
2-Iodohippuric Acid 2-(2-Iodobenzamido)acetic acid; divinylbenzene.
C 9 H sIN0 3,2H zO = 341.1 (147-58-0) Iron Fe = 55.85 (7439-89-6)
White or almost white, crystalline powder, sparingly soluble Grey powder orl wire, soluble in dilute mineral acids.
in water. Iron(m) Chloride, Anhydrous Iron(m) chloride;
• anhydrous ferric chloride; FeCI3 = 162.2 (7705-08-0)
Water (2.5.12) 9% to 13%, determined on 1.000 g. General reagent grade of commerce.
Chromatography Thin-Iayer chromatography (2.2.27), Greenish black crystals or crystalline powder turning orange
using cellulose for chromatography F254 as the coating on exposure to moist airo
substance: apply to the plate 20 ¡.tL of a solution of the Iron(m) Chloride Hexahydrate Ferric chloride, iron
2-iodohippuric acid, prepared by dissolving 40 mg in 4 mL trichloride hexahydrate; FeCI3,6H zO = 270 .3 (10025-77-1)
of 0.1 M sodiumhydroxide and diluting to 10 mL with water. Yellowish-orange or brownish crystalline mas ses,
Develop oyera path of about 12 cm using as the mobile deliquescent, very soluble in water, soluble in ethanol (96%).
phase the upperlayer obtained by shaking together
V-A 76 Appendix 1 A 2014
On exposure to light, ferric chloride and its solutions are alkali hydroxides giving a violet colour becoming yellow on
partly reduced. standing.
Store in an airtight container. Melting point, about 200°, with partial sublimation.
Iron(m) ChIoride Solution Analytical reagent grade of Suljated ash (2.4.14) Maximum 0.2%.
commerce, diluted to contain about 15% w/v of FeCh. Isatin Reagent Dissolve 6 mg of iron(m) sulfate in 8 mL of
Iron(m) ChIoride Solution, Ethanolic Carefully add water and add cautiously 50 mL of sulfurie aeid. Add 6 mg of
25 mL of sulfurie acid dropwise to 75 mL of well-cooled isatin and stir until dissolved. The reagent should be pale
absolute ethanol, stirring constantly. Add 2 g of anhydrous yellow, but not orange or red.
iron(m) ehlonde, stir and filter. Isoamyl Alcohol Amyl alcohol; 3-methylbutan-l-01;
Iron(m) ChIoride Solution Rl Ferric chloride solution C SH 12 0 = 88.1 (123-51-3)
Rl A 10.5% w/v solution of iron(m) ehloride hexahydrate. Colourless liquid, slightly soluble in water, miscible with
Iron(m) ChIoride Solution R2 Ferric chloride solution ethanol (96%).
R2 A 1.3% w/v solution of iron(m) ehlonde hexahydrate. Boiling point, about 130°.
Iron(m) ChIoride-Sulfamic Acid Reagent Ferric Isoamyl Benzoate Isopentyl benzoate; 3-Methylbutyl
chloride-sulfamic acid reagent, iron(m) chloride-sulfamic acid benzoate; C12H1602 = 192.3 (94-46-2)
reagent
n~o about 1.494; boiling point, about 261 °C .
A solution containing 1.0% w/v of iron(m) ehlonde Colourless or pale yellow liquido
hexahydrate and 1.6% w/v of sulfamie aeid.
Isoandrosterone Epiandrosterone; 3 ~-hydroxy- 5 cx
Iron(m) Nitrate Ferric nitrate;
androstan-17-one; C19H3002 = 290.4 (481-29-8)
Fe(N0 3 h,9H 20 = 404.0 (7782-61-8)
White or almost white powder, practically insoluble in water,
Content, minimum 99.0% m/m of Fe(N0 3h,9H 20. soluble in organic solvents.
Light-purple crystals or crystalline mass, very soluble in
[al~o, +88, determined on a 2% w/v solution in methanol.
water.
Free acid Not more than 0.3% (as HN0 3).
M (2.2.41) 14.24 x 10 3 , deterrnined at 304 nm on a
Iron(m) Nitrate Solution A 0.1 % w/v solution of iron(lIl)
0.125% w/vsolution.
nitrate in 0.1 % v/v nitrie acid.
Isohutyl Acetate C 6H 12 0 2 = 116.2 (110-19-0)
Iron Salicylate Solution Dissolve 0.1 g of Jeme
ammonium sulfate in a mixture of 2 mL of dilute sulfurie acid General reagent grade of commerce.
and 48 mL of water and dilute to 100 mL with water. A colourless liquidó boiling point, about 118°; weight per
Add 50 mL of a 11.5 gIL solution of sodium salieylate, mL, about 0.87 g.
10 mL of dilute aeetie acid, 80 mL of a 136 gIL solution of N-Isohutyldodecatetraenamide (2E,4E,8Z, 1OEZ)-N-2-
sodium aeetate and dilute to 500 mL with water. The solution (Methylpropyl)dodeca-2,4,8, 10-tetraenamide;
should be recently prepared. C 16H 2SNO = 247.4 (866602-52-0)
Store in an airtight container, protected from light. White or almost white or non-coloured crystals.
Iron(n) Sulfate Ferrous sulfate; ferrous sulphate; iron(m) Melting point, about 70°.
sulphate; FeS04,7H 20 = 278.0 (7782-63-0) N-Isohutyldodecatetraenamide Solution
Ferrous Sulphate Heptahydrate of the British A solution of N-isobutyldodeeatetraenamide, exactly weighed, in
Pharrnacopoeia. methanol at a concentration of about 10 mg/mL.
Iron(m) Sulfate Ferric sulfate; ferric sulphate; iron(m) Isodrin 1,2,3,4,10,1 O-Hexachloro-l ,4,4a,5,8,8a-hexahydro-
sulphate; iron(m)trisulfate hydrated; iron(m)trisulphate endo,endo-l,4:5,8-dimethanonaphthalene;
hydrated; Fe2(S04)3 + aq (10028-22-5) C 12H s Cl 6 = 364.9 (465-73-6)
Yellowish-white powder, very hygroscopic, decomposes in Practically insoluble in water, soluble in common organic
air, slightly soluble in water and in ethanol (96%). solvents such as acetone.
Store in an airtight container, protected from light. A suitable certified reference solution may be used.
Iron(m) Sulfate Pentahydrate Ferric sulfate Isoleucine Of the British Pharrnacopoeia .
pentahydrate; ferric sulphate pentahydrate; iron(m) sulphate
Isomalt C12H24011 = 344.3 (64519-82-0)
pentahydrate; Fe2(S04h,5H20 = 489.9 (142906-29-4)
Mixture of 6-0-cx-D-glucopyranosyl-D-glucitol and of 1-0-cx-
White or yellowish powder.
D-glucopyranosyl-D-ma=itol.
Iron(n) Sulfate Solution R2 Iron(n) sulphate solution R2
White or almost white powder or granules, freely soluble in
Dissolve 0.45 g of iron(Il) sulfate in 50 mL of water.
O.IM hydrochlon'e acid and dilute to 100 mL with earbon
Isomaltitol 6-0-cx-d-Glucopyranosyl-d-glucitol;
dioxide-free water. Prepare immediately before use.
C1 2H 24011 = 344.3 (534-73-6)
/ Iron(n) Sulfate-Citrate Solution Iron(n) sulphate-citrate
White or almost white powder, freely soluble in water.
solution
Isomenthol (+ )-Isomenthol; (lS,2R,5R)-2-isopropyl-5-
Dissolve 1 g of sodium metabisulfite in 200 mL of water and
methylcyclohexanol; (± )-isomenthol, a mixture of equal
add 0.5 mL of 2M hydroehlon'e aeid, 1.5 g of iron(I1) sulfate
parts of (lS,2R,5R)- and (lR,2S,5S)-2-isopropyl-5-
and 10 g of sodium citrate.
methylcyclohexanol; C lOH 20 0 = 156.3 (23283-97-8)
Colourless crystals, practically insoluble in water, very soluble
Isatin Indoline-2,3-dione; C sH sN0 2 = 147.1 (91-56-5) in ethanol (96%).
Small, yellowish-red crystals, slightly soluble in water, soluble
in hot water and in ethanol (96%), soluble in solutions of
[a]~O,(+)-Isomenthol about + 24, detennined on a 10% w/v d¡O, about 0.911; ng>, about 1.472; boiling point, about 91 °.
solution in ethanol (96%). Isopulegol used in gas chromatography complies with the following
Boiling point, (+)-Isomenthol about 218°; (±)-Isomenthol additional test.
about 218°. Assay Gas chromatography (2.2.28) as prescribed in the
Melting point, (+ )-Isomenthol about 80°; (± )-Isomenthol monograph Mint oil, partly dementholised (1838).
about 53°. Content, minimum 99%, calculated by the normalisation
(+) -Isomenthone (l R)-cis-p- Menthan-3-one; (l R)-cis-2- procedure.
isopropyl-5-methylcyclohexanone; C IO H 18 0 = 154.2 Isoquercitroside Isoquercitrin. 2-(3,4-Dihydroxyphenyl)-
Contains variable amounts of menthone. A colourless liquid, 3-(~-D-glucofuranosyloxy)-5, 7 -dihydtoxy-4H-l-benzopyran-4-
very slightly soluble in water, soluble in ethanol (96%). one, 3,3',4',5,7-Pentahydroxyfiavone-3-glucoside;
d~g, about 0.904; n~o, about 1.453; [a]g>, +93.2. C21H200 12 = 464.4 (21637-25-2)
Isomenthone used in gas chromatography complies with the Isosilibinin 3,5,7 -Trihydroxy-2- [2-( 4-hydtoxy-3-
following additional test. methoxyphenyl) -3-hydroxymethyl-2,3-dihydro-l ,4-
benzodioxin-6-yl] chroman-4-one;
C 2sH 2201O = 482.4 (72581-71-6)
White to yellowish powder, practically insoluble in water,
soluble in acetone and in methano!.
Kaolin, Light (1332-58-7) A purified native hydrated
procedure.
aluminium silicate containing a suitable dispersing agent.
Isomethyleugenol 1,2-Dimethoxy-4-prop-l-enylbenzene;
A grade of commerce complying with the tests for Coarse
CllHl402 = 178.2 (93-16-3)
particles and Fine particles described in the monograph for
Isomethyleugenol used in gas chromatography complies with Light Kaolin.
Kerosene, Deodorised
General grade of commerce.
monograph Niaouli oil, cineole type (2468).
l1-Keto-~-boswellic Acid 3a-Hydroxy-l1-oxours-12-en-
Content Mínimum 97.0%, calculated by the nonnalisation
24-oic acid. (4~) - 3a-Hydroxy-11-oxours-12-en-23-oic acid;
procedure.
C30H4604 = 470 .7 (17019-92-0)
Isoniazid Isonicotinohydrazide; C 6H 7N 30 = 137.1 (54-
White or almost white powder, insoluble in water, soluble in
85-3)
acetone, in anhydrous ethanol and in methano!.
Melting point, 195° to 19T.
Colourless crystals or a white, crystalline powder; melting
11-Keto-p-boswellic acid used in liquid chromatography complies
point, about 171 0 .
Isoniazid Solution Dissolve 0.1 g of isoniazid in 150 mL
Assay Liquid chromatography (2.2.29) as prescribed in the
of methanol, add 0.12 mL of hydrochloric acid and dilute to
monograph on Indian Frankincense.
200 mL with methanol.
Minimum 90%, calculated by the normalisation procedure.
Isonicotinamide C 6H 6N 20 = 122.13 (1453-82-3)
Kieselguhr Acid-purified grade of commerce.
Kieselguhr for Chromatography Kieselguhr complying
Isopentyl Benzoate See Isoamyl benzoate
Filtration rate Use a chromatography column (25 cm x
Isopropylamine Propan-2-amine, 2-propylamine; 10 mm) with a sintered-glass (lOO) plate and two marks at
C3H9N = 59.11 (75-31-0) 10 cm and 20 cm aboye the plate. Place sufficient of the
Colourless, highly volatile, fiammable Iiquid. substance being examined in the column to reach the first
n~o, about 1.374; boiling point, 32° to 34°. mark and fill to the second mark with water. When the first
Isopropyl Iodide 2-Iodopropane; drops begin to fiow from the column, fill to the second mark
C 3H71 = 170.0 (75-30-9) again with water and measure the time required for the first
5 mL to fiow from the column. The fiow rate is not less
Isopropyl Methanesulfonate 1-Methylethyl
than 1 mL per minute. The eluate obtained is colourless.
methanesulphonate; C 4H IO 0 3S = 138.2 (926-06-7)
Kieselguhr G Kieselguhr that has been treated with
Clear, colourless Iiquid.
hydrochloric acid and calcined and to which has been added
Content, minimum 99.0%; density, about 1.129 g cm- 3 about 15 % of calcium sulfate hemihydrate. The average
(20°). particle size is 10 J.lm to 40 J.lm.
ng>, 1.418-1.421; boiling point, about 82° at 6 mm Hg. Complies with the following test.
Isopropyl ~yristate Isopropyl tetradecanoate; Chromatographic separation Carry out the method for
C 17 H 34 0 2 = 270.5 (110-27-0) thin-layer chromatography, Appendix III A, using the
Of the British Phannacopoeia. substance being examined in a 0.27% w/v solution of sodium
4-Isopropylphenol C 9H 120 = 136.2 (99-89-8) acetate to coat the plate and a mixture of 12 volumes of
Content, minimum 98%. water, 23 volumes of propan-2-o1 and 65 volumes of ethyl
acerate as the mobile phase but allowing the solvent front to
Boiling point, about 212°; melting point, 59° to 61 °.
ascend 14 cm aboye the line of application; the development
Isopulegol (-)-Isopulegol; (IR,2S,5R)-2-Isopropenyl-5- time is about 40 minutes. Apply to the plate 5 J.lL of a
methylcyclohexanol; C IOH 18 0 = 154.2 (89-79-2) solution containing 0.01 % w/v of lactose, sucrose, D-glucose and
D-fructose in pyridine. After removal of the plate, allow it to
dry in air and spray with about 10 mL of anisaldehyde solution Temperature:

and heat for 5 10 10 minutes at 100° 10 105°. The
chromatogram shows four well-defined spots without tailing Time Temperature
and well separated from each other. (min) (OC)
Lactic Acid C 3H 60 3 = 90.08 (50-21-5) Column 0-12.5 230 --> 280
Of the British Pharmacopoeia. Inj ection port 250
Lactic Reagent Lactic acid reagent Detector 280
Solution A To 60 mL of lactic acid add 45 mL of
previously filtered lactic acid saturated without heating with
Detection Flame ionisation.
Sudan red G; as lactic acid saturates slowly without heating,
an excess of colorant is always necessary. Inject an appropriate derivatised sample.
Solution B Prepare 10 mL of a saturated solution of Lanatoside C 3B-[(B-D-Glucopyranosyl-(1->4)-3-0-acetyl-
aniline. Filter. 2,6-dideoxy-B-D-ribo-hexopyranosyl-(1->4)-2,6-dideoxy-B-D-
Solution C Dissolve 75 mg of potassium iodide water and nbo-hexopyranosyl-(1-->4)-2,6-dideoxy-B-D-ribo-
dilute 10 70 mL with the same solvento Add 10 mL of hexopyranosyl)oxy]-12B, 14-dihydroxy-5 B-card-20(22)-
ethanol (96%) and 0.1 g of iodine. Shake. enolide; C49H76020 = 985 (17575-22-3)
Mix solutions A and B. Add Solution C. Long flat prisms obtained after recrystallisation in ethanol
(96%).
Lactobionic Acid C 12H 22 0¡2 = 358.3 (96-82-2)
Freely soluble in pyridine and in dioxane.
White or almost white, crystalline powder, freely soluble in
water, practically insoluble in ethanol (96%). Lanthanum ChIoride Heptahydrate
LaCl 3,7H 20 = 371.4
White or almost white powder or colourless crystals, freely
Lactose C¡2H220¡¡,H20 = 360.3 (5989-81-1)
soluble in water.
Lanthanum Chloride Solution To 58.65 g of lanthanum
B-Lactose B-D-Lactose; Cl2H22011 = 342.3 (5965-66-2) m'oxide slowly add 100 mL of hydrochloric acid. Heat to
White or slightly yellowish powder. boiling. Allow to cool and dilute to 1000.0 mL with water.
Content, minimum 99%. Lanthanum Nitrate Lanthanum trinitrate hexahydrate;
ex-D-Iactose, not greater than 35%. La(N0 3h6H 2 0 = 433.0 (10277-43-7)
Assay Gas chromatography (2.2.28): use the normalisation Colourless crystals, deliquescent, freely soluble in water.
procedure. Store in an airtight container.
Column: Lanthanum Nitrate Solution A 5% w/v solution.
- size: 1 = 30 m, 0 = 0.25 mm, Lanthanum Trioxide Lanthanum oxide;
- stationary phase: poly[(cyanopropyl) (phenyl)][dimethyl] La203 = 325.8 (1312-81-8)
siloxane (film thickness 1 Ilm). An almost white, amorphous powder, practically insoluble in
Camer gas helium for chromatography. water. It dissolves in dilute solutions of mineral acids and
Temperature: absorbs atrnospheric carbon dioxide.
Calcium, maximum 5 ppm.
Lauric Acid Dodecanoic acid; Cl 2H2402 = 200.3
Time Temperature
( oC)
(143-07-7)
(min)
Column o-32,S 20 --> 280
White or almost white, crystalline powder, practically
insoluble in water, freely soluble in ethanol (96%).
Injection port 250
Detector 250
Lauric acid used in the assay of total fatty acids in Saw palmetto
fruit (1848) complies with the following additional test.
Detection Flame ionisation. Assay Gas chromatography (2.2.28) as prescribed in the
Inject an appropriate derivatised sample. monograph Saw palmetto fruit (1848).
ex-Lactose Monohydrate ex-D-Lactose monohydrate; Content, minimum 98%, calculated by the normalisation
C¡2H22011,H20 = 360.3 (5989-81-1) procedure.
White or almost white powder. Lauryl Alcohol Dodecan-l-ol;
C l2H 26 0 = 186.3 (112-53-8)
B-D-Iactose, less than 3%.
dgg, about 0.820; melting point, 24° 10 27".
Content, minimum 98.0%, determined by gas
Assay Gas chromatography (2. 2.28): use the normalisation
chromatography.
procedure.
Lavandulol 2-Isopropenyl-5-methyIhex-4-en-l-0I, (R)-5-
Column:
methyl-2-(1-methylethenyl)-4-hexen-l-ol;
- size: 1= 30 m, 0 == 0.25 mm, ClQH 1S O = 154.2 (498-16-8)
- stationary pha,se: poly(dimethyl)siloxane (film thickness Oily liquid with a characteristic odour,
1 Ilm).
Lavandulol used in gas chromatography complies with the
Camer gas helium for chromatography. following additional test.
monograph Lavender oil (1338).
Test solution The substance to be examined. White or almost white powder, soluble in water, freely
Content, minimum 90.0%, calculated by the normalisation soluble in methanol, slightly soluble in ethanol (96%).
procedure. Melting point, 190 c to 193".
Lavandulyl Acetate 2-Isopropenyl-5-methylhex-4-en-l-yl Lemon Oil Of the British Pharmacopoeia.
acetate; C12Hzo02 = 196.3 (25905-14-0) Leucine (61-90-5) Of the British Pharmacopoeia.
Colourless liquid with a characteristic odour. L-Leucine See leucine.
Lavandulyl acetace used in gas chromatography complies with the Levodopa (59-92-7) Of the British Pharmacopoeia.
following additional test. Levomenol See (-)-fJ.-bisabolol.
Assay Gas chromatography (2.2.28) as prescribed in the (Z)-Ligustilide (3Z)-3-Butylidene-l,3,4,5-
monograph Lavender oil (J 338). tetrahydroisobenzofuran-l-one;
Test solution The substance to be examined. C12H1402 = 190.2 (81944-09-4)
Content, mínimum 93.0%, calculated by the normalisation General reagent grade of commerce.
procedure. Limonene D-Limonene; (+) -p-mentha-l,8-diene; (R)-4-
Lead Acetate Lead(u) aceta te, lead di-acetate; isopropenyl-l-methylcyclohex-l-ene;
C4 H 6 0 4 Pb,3HzO = 379.3 (6080-56-4) ClOH1 6 = 136.2 (5989-27-5)
Colourless crystals, effiorescent, freely soluble in water, Colourless liquid, practically insoluble in water, soluble in
soluble in ethanol (96%). ethanol (96%).
Lead(n) Acetate See lead acetate. dig, about 0.84; nf,C', 1.471 to 1.474; [Cl'l~o, about + 124;
Lead Acetate eotton Immerse absorbent cotton in a boiling point, 175° to 177°.
mixture of 1 volume of dilute acetic acid and 10 volumes of Limonene used in gas chromatography complies with ¡he following
lead acetate solution. Drain off the excess of liquid, without additional test.
squeezing the cotton, by placing it on several layers of filter Assay Gas chromatography (2.2.28) as prescribed in the
paper. Allow to dry in air. monograph Peppermint oi! (0405).
Store in an airtight container. Test solution The substance to be examÍned.
Lead Acetate Paper Immerse filter paper weighing about Content, minimum 99.0%, calculated by the normalisation
80 g/m z in a mixture of 1 volume of dilute acetic acid and procedure.
10 volumes of lead acetate solutúm. After drying, cut the
LinaIol (RS)- 3,7 -Dimethylocta-l ,6-dien-3-01, linalool;
paper into strips 15 mm by 40 mm.
ClOH1 SO = 154.2 (78-70-6)
Lead Acetate Solution A 9.5 % w/v solution in carbon
Mixture of two stereoisomers (licareol and coriandrol).
dioxide-free water.
Liquid, practically insoluble in water.
Lead Dioxide Lead(Iv) oxide; Pb0 2 = 239.2 (1309-60-0)
Dark brown powder, evolving oxygen when heated, dig, c about 0.860; nf,C', about 1.462; boiling point, about
practically insoluble in water, soluble in hydrochloric acid 200 •
with evolution of chlorine, soluble in dilute nitric acid in the Linalol used in gas chromatography complies with the following
presence of hydrogen peroxide, oxalic acid or other reducing test.
agents, soluble inlhot, concentrated alkali hydroxide Assay Gas chromatography (2.2.28) as prescribed in the
solutions. monograph Anise oil (0804).
Lead Nitrate Lead dinitrate, lead(u) nitra te; Test solution The substance to be examined.
Pb(N0 3)z = 331.2 (10099-74-8) Content, minimum 98.0%, calculated by the normalisation
White or almost white, crystalline powder or colourless procedure.
crystals, freely soluble in water. LinaIool See linalol.
Lead(n) Nitrate See lead nitrace. LinaIyl Acetate (RS)-1,5-Dimethyl-l-vinylhex-4-enyl
Lead Nitrate Solution A 3.3% w/v solution. acetate; ClzHzo02 = 196.3 (115-95-7)
Lead(Iv) Oxide See lead dioxide. Colourless or slightly yellow liquid with a strong odour of
Lead Subacetate Solution Basic lead acetate solution bergamot and lavender.
(1335-32-6) di~, 0.895 to 0.912; n~o, 1.448 to 1.451; boiling point, about
Content, 16.7% m/m to 17.4% m/m ofPb (Ar 207.2) in a 215°.
form corresponding approximately to the formula Linalyl acetate used in gas chromatography complies with the
C SH 14 0 lO Pb 3 · following additional test.
Dissolve 40.0 g of lead acetate in 90 mL of carbon dioxide-free Assay Gas chromatography (2.2.28) as prescribed in the
water. Adjust the pH to 7.5 with strong sodium hydroxide monograph Bitter-orange-fiower oi! (J 175).
solution. Centrifuge and use the clear colourless supernatant Test solution The substance to be examined.
solution. Content, mínimum 95.0%, calculated by the normalisation
The solution remains clear when stored in a well-closed procedure.
container. Lindane y- Hexachlorocyc1ohexane;
Leiocarposide 2-W-D-Glucopyranosyloxy)benzyl 3-W-D- C6H6Cl6 = 290.8 (58-89-9)
glucopyranosyloxy)-6-hydroxy-2-methoxybenzoate; 2-[[[3-W- For the monograph Wool fat (0134), a suitable certified
D-Glucopyranosyloxy)-6-hydroxy-2- reference solution (lO ng/IlL Ín cyc1ohexane) may be used.
methoxybenzoyl) oxy) methyl) phenyl-~-D-glucopyrano side;
Linoleic Acid (9Z,12Z)-Octadeca-9,12-dienoic acid;
C27H34016 = 614.5 (71953-77-0)
CI sH3Z0Z = 280.5 (60-33-3)
V-ASO Appendix 1 A 2014
Colourless, oily liquid. Lithium Hydroxide Lithium hydroxide monohydrate;

d¡O, about 0.903; n~, about 1.470. LiOH,H 20 = 41.96 (1310-66-3)
Linoleic acid used in the assay of total fatly acids in Saw White or almost white, granular powder, strongly alkaline, it
Palmetto Fruit complies with the following additional test. rapidly absorbs water and carbon dioxide, soluble in water,
sparingly soluble in ethanol (96%).
monograph Saw palmetto fruit (1848). Store in an airtight container.
Content, minimum 98%, calculated by the normalisation Lithium Metaborate, Anhydrous
procedure. LiB0 2 = 49.75 (13453-69-5)
Linolenic Acid (9Z, 12Z,15Z)-Octadeca-9, 12, 15-trienoic General reagent grade of commerce.
acid; CIsH3002 = 278.4 (463-40-1) Lithium Sulfate Dilithium sulfate monohydrate; Lithium
Colourless liquid, practically insoluble in water, soluble in sulphate; Li 2S0 4,H 2 0 = 128.0 (10102-25-7)
organic solvents. Colourless crystals, freely soluble in water, practically
d¡O, about 0.915; n~o, about 1.480. insoluble in ethanol (96%).
Linolenic acid used in the assay of total fatly acids in Saw Lithium trifluoromethanesulfonate Lithium
Palmetto Fruit complies with the following additional test. trifiuoromethanesulphonate;
CF3 Li0 3 S = 156.0 (33454-82-9)
monograph Saw palmetto fruit (1848). Litmus (1393-92-6)
Content, minimum 98%, calculated by the normalisation Indigo-blue fragments prepared from various species of
procedure. Rocella, Lecanora or other lichens, soluble in water,
practically insoluble in ethanol (96%).
Linolenyl Alcohol (9Z, 12Z, 15Z)-octadeca-9, 12, 15-trien-l-
01; C 1sH 32 0 = 264.4 (24149-05-1) Colour change pH 5 (red) to pH 8 (blue).
Content, minimum 96%. Litmus Paper Use red litmus paper or blue litmus paper, as
appropriate.
Linoleyl Alcohol (9Z, 12Z)-octadeca-9, 12-dien-l-01;
C 1sH 340 = 266.5 (506-43-4) Litmus Paper, Blue Boil 10 parts of coarsely powdered
litmus for 1 hour with 100 parts of ethanol (96%). Decant the
Relative density, 0.830.
alcohol and add to the residue a mixture of 45 parts of
Content, minimum 85%. ethanol (96%) and 55 parts of water. After 2 days decant the
Linsidornine Hydroch1oride 3-(Morpholin-4- clear liquido Impregnate strips of filter paper with the
yl)sydnonimine hydrochloride; 3-(Morpholin-4-yl)-1,2,3- solution and allow to dry.
oxadiazol-3-ium-5-aminide hydrochloride; Test/or sensitivity Immerse a strip measuring 10 mm by
C6HIICIN402 = 206.6 (16142-27-1) 60 mm in a mixture of 10 mL of 0.02 M hydrochloric acid
White or almost white powder. and 90 mL of water. On shaking the paper turns red within
Lipase Solvent See Maleate Buffer Solution pH 7.0. 45 seconds.
Liquid Scintillation Cocktail Litmus Paper, Red
Commercially available solution for the determination of To the blue litmus extract, add dilute hydrochloric acid
radioactivity by liquid scintillation counting. It contains one dropwise until the blue colour becomes red. Impregnate
or more fiuorescent agents and mostly on~ or more strips of filter paper with the solution and allow to dry.
emulsifying agents in a suitable organic solvent or mixture of Test/or sensitivity Immerse a strip measuring 10 mm by
organic solvents. 60 mm in a mixture of 10 mL of 0.02 M sodium hydroxide
Liquid Scintillation Cocktail Rl To 1000 mL of dioxan, and 90 mL of water. On shaking the paper tums blue within
add 0.3 g of methylphenyloxazolylbenzene, 7 g of 45 seconds.
diphenyloxazole and 100 g of naphthalene. Litmus Solution Boil 25 g of coarsely powdered litmus
Lithium Li = 6.94 (7439-93-2) with 100 mL of ethanol (90%) under a refiux condenser for
A soft metal whose freshly cut surface is silvery-grey. 1 hour, discard the clear liquid and repeat this operation
It rapidly tamishes in contact with air. It reacts violently with using two 75-mL quantities of ethanol (90%). Digest the
water, yielding hydrogen and giving a solution of lithium extracted litmus with 250 mL of water and filter.
hydroxide; soluble in methanol, yielding hydrogen and a Loganin Methyl (IS,4aS,6S,7R,7aS)-I-(~-D
solution of lithium methoxide; practically insoluble in light glucopyranosyloxy)-6-hydroxy-7-methyl-l,4a,5,6, 7, 7a-
petroleum. hexahydrocyclopental[c]pyran-4-carboxylate;
Store under light petroleum or liquid paraffin. C 17H 260 IO = 390.4 (18524-94-2)
Lithium and Sodium Molybdotungstophosphate Melting point, 220° to 221 °.
Solution See Phosphomolybdotugstic reagent. Longifolene (IS,3aR,4S,8aS)-4,8,8- Trimethyl-9-
Lithium Carbonate Dilithium carbonate; methylenedecahydro-l,4-methanoazulene;
Li 2C0 3 = 73.9 (554-13-2) C 1sH 24 = 204.4 (475-20-7)
White or almost white, light powder, sparingly soluble in Oily, colourless liquid, practically insoluble in water, miscible
water, very slightly soluble in ethanol (96%). A saturated with ethanol (96%).
solution at 20° contains about 13 gIL of Li2C03. dl s, 0.9319; n~, 1.5050; [a]~, + 42.7; boiling point, 254° to
Lithium Ch10ride LiCI = 42.39 (7447-41-8) 256°.
Crystalline powder or granules or cubic crystals, deliquescent, Longifolene used in gas chromatography complies with the
freely soluble in water, soluble in acetone and in ethanol following additional test.
(96%). Aqueous solutions are neutral or slightly alkaline. Assay Gas chromatography (2.2.28) as prescribed in the
Store in an airtight container. monograph Turpentine oil) Pinus pinaster type (1627) .
Content, minimum 98.0%, calculated by the normalisation Arsenic (2.4.2, Method A) Maximum 2 ppm.
procedure. Dissolve 0.5 g in a mixture of 5 mL of water and 5 mL of
Low-vapour-pressure hydrocarbons (type L) hydroehlorie acid R1.
Unctuous mass, soluble in benzene and in toluene. Heavy metals (2.4.8) Maximum 10 ppm .
Lumiflavine 7,8,10-Trimethylbenzol [g]pteridine- Dissolve 1.0 g in a mixture of 3 mL of water and 7 mL of
2,4(3H,10H)-dione; C 13H 12 N 4 0 2 = 256.3 (1088-56-8) hydroehlorie aeid R1. Add 0.05 mL of phenolphthalein solution
Yellow powder or orange crystals, very slightly soluble in and eoneentrated ammonia until a pink colour is obtained.
water, freely soluble in methylene chloride. Neutralise the excess of ammonia by the addition of glacial
Luteolin-7-glucoside 2-(3,4-Dihydroxyphenyl)-7-(~-D aeetie acid. Add 0.5 mL in excess and dilute to 20 mL with
glucopyranosyloxy)-5-hydroxy-4H-1-benzopyran-4-one; water. Filter, if necessary. 12 mL of the solution complies
with test A. Prepare the reference solution using a mixture of
C21H20011 = 448.4 (5373-11-5)
5 mL of lead standard solution (I ppm Pb) and 5 mL of water.
Yellow powder.
[ron (2.4.9) Maximum 50 ppm.
Absorbance (2.2.25). A solution in methanol shows
Dissolve 0.2 g in 6 mL of dilute hydroehlorie acid and dilute to
absorption maxima at 255 nm, 267 nm, 290 nm and
10 mL with water.
350 nm.
Melting point, about 247°. Magnesium Silicate for Pesticide Residue Analysis
(1343-88-0)
Macrogol 23 Lauryl Ether Of the British Pharmacopoeia.
The number of moles of ethylene oxide reacted per mole of Magnesium silicate for chromatography (60-100 mesh) .
lauryl alcohol being 23 (nominal value). Magnesium Sulfate Magnesium sulphate;
MgS0 4 ,7H 20 = 246.8 (10034-99-8)
Macrogol200 See Polyethylene glyeol 200.
Macrogol200 Rl See Polyethylene glyeol 200 R1 . Magnesium Sulfate Heptahydrate of the British
Pharmacopoeia.
Macrogol300 See Polyethylene glyeol 300.
Magneson Azo violet; 4-( 4-nitrophenylazo )resorcinol;
Macrogol 400 See Polyethylene glyeol 400. C 12H g N 3 0 4 = 259.2 (74-39-5)
Macrogoll000 See Polyethylene glyeol JOOO. lndicator grade of commerce.
Macrogol 1500 See Polyethylene glyeol 1500. When used for titration in non-aqueous media, it changes
Macrogol20,000 See Polyethylene glyeol 20,000. from orange (acidic) through pink (neutral) to blue (basic).
Macrogol 20,000 2-Nitroterephthalate See Polyethylene Magneson Reagent A 0.1 % w/v solution of magneson in a
glyeol 20,000 2-nitroterephthalate. 1% w/v solution of sodium hydroxide.
Magnesium Mg = 24.30 (7439-95-4) Magneson Solution A 0.2 % w/v solution of magneson in
Silver-white ribbon, turnings or wire, or a grey powder. toluene.
Magnesium Acetate Magnesium diacetate tetrahydrate; Magnolol 5,5 ' -Di(prop-2-enyl)biphenyl-2,2 ' -diol,
C 4H 6Mg0 4,4H 20 = 214.5 (16674-78-5) 5,5'-Diallyl-2,2'-dihydroxybiphenyl, 5,5'-Di-2-propenyl-
Colourless crystals, deliquescent, freely soluble in water and [l,l'-biphenyl]-2,2'-diol; ClsHl S02 = 266.3 (528-43-8)
in ethanol (96%). Maize Oil Refined Maize Oil of the British
Store in an airtight container. Pharmacopoeia.
Magnesium Chloride MgCI 2,6H 20 = 203.3 (7791-18-6) Malachite Green Cl 42000; basic green 4;
Magnesium Chloride Hexahydrate of the BP. C23H2SCIN2 = 364.9 (123333-61-9)
Green crystals with a metallic lustre, very soluble in water
Magnesium Nitrate Magnesium nitrate hexahydrate;
giving a bluish-green solution, soluble in ethanol (96%) and
Mg(N0 3h, 6H20 = 256.4 (13446-18-9)
in methanol.
Colourless, clear crystals, deliquescent, very soluble in water,
Absorbance (2.2.25) A 0.01 gIL solution in ethanol (96%)
freely soluble in ethanol (96%).
shows an absorption maximum at 617 nm.
Malachite Green Solution A 0.5% w/v solution in
Magnesium Nitrate Solution Dissolve 17.3 g of anhydrous aeetie aeid.
magnesium nitrate in 5 mL of water warming gently and add
Malathion ClOH1906PS2 = 330.3 (121-75-5)
80 mL of ethanol (96%). Cool and dilute to 100 mL with
the same solvent. Boiling point, about 156°.
Magnesium Nitrate Solution Rl Dissolve 20 g of A suitable certified reference solution (1 O ng/~IL in iso-
magnesium nitra te (Mg(N0 3)2,6H 20) in deionised distilled octane) may be used.
water and dilute to 100 mL with the saine solvent. Maleic Acid (Z)-But-2-ene-1,4-dioic acid;
lmmediately before use, dilute 10 mL to 100 mL with C4 H 4 0 4 = 116.1 (110-16-7)
deionised distilled water. A volume of 5 ¡.¡L will provide Of the British Pharmacopoeia.
0.06 mg of Mg (N0 3h Maleic Anhydride Furan-2,5-dione;
Magnesium Oxide Light magnesium oxide; C 4 H 20 3 = 98.06 (108-31-6)
MgO = 40.31 (1309-48-4) White or almost white crystals, soluble in water forming
Light Magnesium Oxide of the British Pharmacopoeia. maleic acid, very soluble in acetone and in ethyl aceta te,
Magnesium Oxide, Heavy freely soluble in toluene, soluble in ethanol (96%) with ester
Heavy Magnesium Oxide of the British Pharmacopoeia. formation, very slightly soluble in light petroleum.
Magnesium Oxide Rl MgO = 40.31 Melting point, about 52°.
Complies with the requirements prescribed for magnesium Any residue insoluble in toluene do es not exceed 5% (maleic
oxide with the following modifications. acid).
Maleic Anhydride Solution Dissolve 5 g of maleic Menadione 2-Methyl-l,4-naphthoquinone;

anhydride in toluene and dilute to 100 mL with the same CllHsOz = 172.2 (58-27-5)
solvent. Use within one month. If the solution becomes Of the British Pharmacopoeia.
turbid, filter. Menthofuran 3,9-Epoxy-p-mentha-3,8-diene; 3,6-
Mallc Acid DL-Hydtoxysuccinic acid; dimethyl-4,5,6,7-tetrahydrobenzofuran;
C 4H 7 0 S = 134.1 (617-48-1) C lO H 140 = 150.2 (17957-94-7)
General reagent grade of commerce. Slightly bluish liquid, very slightly soluble in water, soluble in
Melting point, about 129°. ethanol (96%).
Maltitol (585-88-6) dr~, about 0.965; n;o, about 1.480; [a];o, about +93; boiling
Of the British Pharmacopoeia. point, 196°.
Maltotriose a-D-Glucopyranosyl-(1->4)-a-D- Menthofuran used in gas chromatography complies with the
glucopyranosyl-(1->4)-D-glucose; following additional test.
ClsH32016 = 504.4 (1109-28-0) Assay Gas chromatography (2.2.28) as prescribed in the
Mandelic Acid 2-Hydroxy-2-phenylacetic acid; monograph Peppermint oil (0405).
C SH S0 3 = 152.1 (90-64-2) Test solution The substance to be exarnined.
White crystalline fiakes, soluble in water; melting point, 118° Content Mínimum 97.0%, calculated by the normalisation
to 121 °. procedure.
Manganese(lv) Oxide, Pre-washed Manganese(IV) Menthol (-)-p-Menthan-3-01 (2216-51-5); (±)-p-menthan-
Oxide Activated; Mn0 2 = 86.9 (1313-13-9) 3-01 (15356-70-4) ; CIOHzoO = 156.3
General reagent grade of commerce. Levomenthol and Racemic Menthol of the British
Melting point, about 535°. Pharmacopoeia.
Manganese Sulfate See Manganese(JI) sulfate. Menthol used in gas chromatography complies with the following
additional test.
Manganese(n) Sulfate Manganese sulfate; manganese
sulphate; manganese(n) sulphate; Assay Gas chromatography (2.2.28) as prescribed in the
MnS0 4,H 20 = 169.0 (10034-96-5) related substances test inc1uded in the monograph Racemic
menthol (0623).
Pale-pink, crystalline powder or crystals, freely soluble in
water, practically insoluble in ethanol (96%). Content Mínimum 98.0%, calculated by the normalisation
procedure.
Loss on ignition 10.0% to 12.0%, determined on1.000 g
at 500 ± 50°. Menthone (-)-trans-p-Menthan-3-one; (2S,5R)-2-
isopropyl-5-methylcyc1ohexanone;
Mannitol See o-Mannitol.
ClOH1SO = 154.2 (14073-97-3)
D-Mannitol Mannitol; C 6H 140 6 = 182.2 (69-65-8)
Contains variable amounts of isomenthone.
Mannitol of the British Pharmacopoiea.
Colourless liquid, very slightly soluble in water, very soluble
Mannose See D-Mannose. in ethanol (96%) .
D-Mannose Mannose; C 6H 12 0 6 = 180.2 (3458-28-4) d~g, about 0.897; n;o, about 1.450.
White or almost white, crystallíne powder or small crystals, Menthone used in gas chromatography complies with the following
very soluble in water, slightly soluble in anhydrous ethano!. additional test.
[a];o, + 13.7 + 14.7, determined on a 200 gIL solution in Assay Gas chromatography (2.2.28) as prescribed in the
water R containíng about 0.05% ofNH 3. monograph Peppermint oil (0405).
Melting point, about 132°, with decomposition. Test solution The substance to be examined.
Marrubiin (2aS,5aS,6R, 7R ,8aR,8bR)-6-[2-(Furan- 3- Content Minimum 90.0%, calculated by the normalisation
yl)ethyl]-6-hydroxy-2a,5a, 7-trimethyldecahydro-2H- procedure.
naphtho[I,8-bc]furan-2-one; C2oH 2S04 = 332.4 (465-92-9)
Menthyl Acetate ClzH220 2 = 198.3 (2623-23-6)
Colourless, microcrystalline powder.
Colourless liquid, slightly soluble in water, miscible with
Marrubiin used in liquid chromatography complies with the ethanol (96%).
following additional test.
4g, about 0.92; n;o, about 1.447; boiling point, about 228°.
Menthyl acetate used in gas chromatography complies with the
monograph on White horehound.
Content Mínimum 95.0%, calculated by the normalisation
procedure. Assay Gas chromatography (2.2.28) as prescribed in the
Mebendazole C16H13N303 = 295.3 (31431-39-7)
Test solution. The substance to be exarnined.
Content Mínimum 97.0%, calculated by the normalisation
Meclozine Hydrochloride Mec10zine dihydrochloride; procedure.
C 2s H 27 CIN2,2HCI = 463 .9 (1104-22-9)
Mercaptoacetic Acid Thioglycollic acid;
Mec10zine Dihydrochloride of the British Pharmacopoeia. C 2H 40 ZS = 92.12 (68-11-1)
Melamine 1,3,5-Triazíne-2,4,6-triamine; General reagent grade of commerce.
C3H6N6 = 126.1 (108- 78-1)
A colourless liquidó weight per mL, about 1.33 g.
A white or almost white, amorphous powder, very slightly
soluble in water and in ethanol (96%). 2-Mercaptobenzimidazole lH-benzimidazole-2-thiol;
C7H6NZS = 150.2 (583-39-1)
0
2014 Appendix 1 A V -AS3
2-Mercaptoethanol C 2H 6 0S = 78.1 (60-24-2) Mercury(n) Iodide Mercuric iodide, mercury di-iodide;

Liquid, miscible with water. HgI 2 = 454.4 (7774-29-0)
d~g, about l.l16; boiling point, about 157 .
c Dense, scarlet, crystalline powder, slightly soluble in water,
sparingly soluble in acetone and in ethanol (96%), soluble in
Mercaptopurine See 6-Mercaptopurine.
an excess of potassium iodide solution.
6-Mercaptopurine Mercaptopurine; purine-6-thiol;
C SH 4 N 4 S,H 20 = 170.2 (6112-76-1)
Mercury(u) Nitrate Mercuric nitra te, mercury dinitrate
Mercaptopurine of the Brtish Pharmacopoeia.
monohydrate; Hg(N0 3)z,H 20 = 342.6 (7782-86-7)
Mercuric Acetate See Mercury(n) acetate.
Colourless or slightly coloured crystals, hygroscopic, soluble
Mercuric Acetate Solution See M ercury(n) acetate in water in the presence of a small quantity of nitric acid.
solution.
Store in an airtight container protected from light.
Mercuric Bromide See Mercury(lI) bromide.
Mercury, Nitric Acid SolutÍon of
Mercuric Bromide Paper See Mercury(llj bromide papero
Carefully dissolve 3 mL of mercury in 27 mL ofjwning nitl1C
Mercuric Chloride See Mercury(lI) chloride. acid. Dilute the solution with an equal volume of water.
Mercuric Chloride SolutÍon See Mercury(llj chloride Store protected from light, use within 2 months.
solution.
Mercury(n) Oxide, Yellow Mercuric oxide, mercury
Mercuric Iodide See Mercury(n) iodide. oxide; HgO = 216.6 (21908-53-2)
Mercuric Nitrate See Mercury(llj nitrate. A yellow to orange-yellow powder, practically insoluble in
Mercuric Oxide See Yellow mercury(lI) oxide. water and in ethanol (96 %).
Mercuric Sulfate Solution See Mercury(llj sulfate solution. Store protected from light.
Mercuric Thiocyanate See M ercury(lI) thiocyanate. Mercury(u) Sulfate Solution Mercuric sulfate solution,
Mercuric Thiocyanate SolutÍon See Mercury(llj mercuric sulphate solution, mercury(n) sulphate solution
thiocyanate solution. Dissolve 1 g of mercuric oxide in a mixture of 20 mL of water
Mercury Hg = 200.6 (7439-97-6) and 4 mL of sulfuric acid.
Silver-white liquid, breaking into spherical globules which do Mercury(n) Thiocyanate Mercuric thiocyanate, mercury
not leave a metallic trace when rubbed on paper. di(thiocyanate); Hg(SCN)2 = 316 .8 (592-85-8)
d~g, about 13.5; boiling point, about 357°. White or almost wrute, crystalline powder, very slightly
Mercury(n) Acetate Mercuric aceta te, mercury diacetate; soluble in water, slightly soluble in ethanol (96%), soluble in
C 4H 6 Hg0 4 = 318.7 (1600-27-7) solutions of sodium chloride.
White or almost white crystals, freely soluble in water, Mercury(n) Thiocyanate SolutÍon Mercuric thiocyanate
soluble in ethanol (96 %). solution
Mercury(u) Acetate SolutÍon Mercuric acetate solution Dissolve 0.3 g of mercuric thiocyanate in anhydrous ethanol and
dilute to 100 mL with the same solvento
Dissolve 3.19 g of mercuric acetate in anhydrous acetic acid and
dilute to 100 mL with the same acid. If necessary, neutralise Use within 1 week.
the solution with 0.1 M perchloric acid using 0.05 mL of Mesityl0xide 4-Methylpent-3-en-2-one;
crystal violet solution as indicator. C 6H IO O = 98.1 (141-79-7)
Mercury(n) Bromide Mercuric bromide, mercury Colourless, oily liquid, soluble in 30 parts of water, miscible
dibromide; HgBr2 = 360.4 (7789-47-1) with most organic solvents.
d~g , about 0.858; boiling point, 129 to 130
0 0
White or faintly yellow crystals or a crystalline powder, •
slightly soluble in water, soluble in ethanol (96%). Metanil Yellow CI 13065; 4 '-anilinoazobenzene-3-sulfonic
Mercury(n) Bromide Paper Mercuric bromide paper acid sodium salt; Cl sHl 4N3Na03S = 375 .4 (587-98-4)
In a rectangular dish place a 50 giL solution of mercuric A brownish-yellow powder, soluble in water and in ethanol
bromide in anhydrous ethanol and immerse in it pie ces of white (96%).
filter paper weighing 80 g per square metre (speed of Metanil Yellow Solution A 0.1 % w/v solution in
filtration = filtration time expressed in seconds for 100 mL methanol.
of water at 20 with a filter surface of 10 cm 2 and constant
0
Testfor sensitivity To 50 mL of anhydrous acetic acid

pressure of 6.7 kPa: 40 seconds to 60 seconds), each add 0.1 mL of the metanil yellow solution. Add 0.05 mL of
measuring 1.5 cm by 20 cm and folded in two. Allow the 0.1 M perchloric acid; the colour changes from pinkish-red to
excess liquid to drain and allow the paper to dry, protected violeto
from light, suspended over a non-metallic thread. Discard
Colour change pH 1.2 (red) to pH 2.3 (orange-yellow).
1 cm from each end of each strip and cut the remainder into
1.5 cm squares or discs of 1.5 cm diameter. Metaphosphoric Acid (HP0 3)x (37267-86-0)
Store in a glass-stoppered container wrapped with black Glassy lumps or sticks containing a proportion of sodium
paper. metaphosphate, hygroscopic, very soluble in water.
Mercury(n) Chloride Mercuric chloride; Nitrates Boil1.0 g with 10mL of water, cool, add 1 mL of
HgCl 2 = 271.5 (7487-94-7) indigo carmine solution, 10 mL of nitrogen-free sulphuric acid
and heat to boiling. The blue colour is not entirely
Mercuric Chloride of the British Pharmacopoeia.
discharged.
Mercury(u) Chloride SolutÍon Mercuric chloride
Reducing substances Maximum 0.01 %, ca\culated as
solution
H 3 P0 3 ·
A 5.4 % w/v solution.
Dissolve 35.0 g in 50 mL of water. Add 5 mL of a 200 gIL Methanol, Hydrochloric Dilute 1.0 mL of hydrochlon·c
solution of sulfuric acid, 50 mg of potassium bromide and acid R1 to 100.0 mL with methanol.
5.0 mL of 0.02 M potassium bromate and heat on a water- Methanol Rl
bath for 30 minutes. Allow to cool and add 0.5 g of Complies with the requirements prescribed for methanol and
potassium iodide. Titrate the liberated iodine with 0.1 M the following additional requirement.
sodium thiosulfate, using 1 mL of starch solution as indicator.
Carry out a blank test. Minimum transmittance (2.2.25) using water as
compensation liquid : 20% at 210 nm, 50% at 220 nm, 75%
1 mL of 0.02 M potassium bromate is equivalent to 4.10 mg of at 230 nm, 95% at 250 nm, 98% at 260 nm and at higher
H 3 P0 3 · wavelengths.
Store in an airtight container. Methanol R2
Methacrylic Acid 2-Methylprop-2-enoic acid; Complies with the requirements prescribed for methanol and
C 4 H ó0 2 = 86.1 (79-41-4) the following additional requirements.
Colourless liquid. Content, minimum 99.8%.
n~, about 1.431; boiling point, about 160°; melting point,
Absorbance (2.2.25) Maximum 0.17, deterrnined at
about 16°. 225 nm using water as the compensation liquido
Methane CH 4 = 16 (74-82-8) Methimazole See Thiamazole
Content, minimum 99.0% v/v. DL-Methionine C SH 11 N0 2S = 149.2 (59-51-8)
Methane Rl CH4 = 16 (74-82-8) Of the British Pharrnacopoeia.
Content, minimum 99.995 % v/v. L-Methionine C SH Il N0 2 S = 149 .2 (63-68-3)
Methanesulfonic Acid Methanesulphonic acid; Of the British Pharrnacopoeia.
CH40 3S = 96.10 (75-75-2)
(RS) -Methotrexate (RS)- 2-[4-[[ (2,4-diaminopteridin-6-
Clear colourless liquid, solidifying at about 20°, miscible yl)methyl] methylamino] benzoylamino]pentanedioic acid;
with ~ater, slightly soluble in toluene, practically insoluble in
C2oH 22N sOs = 454.4 (60388-53-6)
. hexane.
d~g, about 1.48; n~, about 1.430.
Methanesulfonic Acid, Methanolic Methanesulphonic
acid, methanolic Methoxyazobenzene C 13H I2NO = 212.2 (2396-60-3)
Solutions of the requisite molarity may be obtained by Use a grade of commerce suitable for chromatographic
dissolving the appropriate quantity of methanesulfonic acid in separations.
methanol. M elting point, about 55°.
Methanesulfonyl Chloride Methanesulphonyl chloride; Methoxychlor 1, 1-(2,2,2-Trichloroethylidene)-
CH3Cl0 2S = 114.6 (124-63-0) bis (4-methoxybenzene); ClóHIsCl30 2 = 345.7 (72-43-5)
Clear, colourless or slightly yellow liquid. Practically insoluble in water, freely soluble in most organic
Content, minimum 99 .0%. solvents.
D ensity, 1.48 g/cm 3. Boiling point, about 346°; melting point, 78° to 86°.
n~o, about 1.452; boiling point, about 161°. A suitable certified reference solution (10 ng/¡,¡L in iso-
octane) may be used.
Methanol Methyl alcohol; CH 4 0 = 32.04 (67-56-1)
trans-2-Methoxycinnamaldehyde
Clear, colourless, flammable liquid, miscible with water and
CIQH 10 0 2 = 162.2 (60125-24-8)
with ethanol (96%) .
d~g, 0.791 to 0.793; boiling point, 64° to 65°.
trans-2-Methoxycinnamaldehyde used in gas chromatography
When 'methano? is followed by a percentage figure, an
camplies with the following additional test.
instruction to use methanol diluted with water to produce the
specified percentage v/v of methanol is implied. Assay Carry out the method for Chromatographic profile
described in the monograph for Cassia Oil using the reagent
Methanol, Acidified To 900 mL of methanol add 18 mL
being examined. The content is not less than 96 .0%, by
of glacial acetic acid and dilute to 1000 mL with water.
normalisation.
Methanol, Aldehyde-free
2-Methoxyethanol Ethylene glycol monomethyl ether;
Dissolve 25 g of iodine in 1 L of methanol and pour the C 3H s0 2 = 76.10 (109-86-4)
solution, with constant stirring, into 400 mL of 1 M sodium
hydroxide. Add 150 mL of water and allow to stand for
16 hours. Filter. Boil under a reflux condenser until the A colourless liquid; boiling point, about 125°; d~g, about
odour of iodoforrn disappears. Distil the solution by 0.93; n~, about 1.406 .
fractional distillation. (IRS) -1-( 6-Methoxynaphthalen-2-yl)ethanol
Aldehydes and ketones Maximum 0.001 %. 6-Methoxy-ex-methyl-2-naphthalenemethanol;
C 13 H I4 0 2 = 202 .3 (77301-42-9)
Methanol, Anhydrous
White or almost white powder.
Treat 1000 mL of methanol with 5 g of magnesium. If
necessary initiate the reaction by adding 0.1 mL of mercuric Melting point, about 113°.
chloride solution. When the evolution of gas has ceased, distil 1-(6-Methoxynaphthalen-2-yl)ethanone 6'-Methoxy-2'-
the liquid and collect the distillate in a dry container acetonaphthone; C 13 H 120 2 = 200.2 (3900-45-6)
protected from moisture. White or almost white powder.
Water (2.5.12) Maximum 0.3 gIL. Melting point, about 108°.
6-Methoxy-2-naphthoic acid 6-Methoxynaphthalene-2- Test solution The substance to be examined.

carboxylic acid; C 12H lO 0 3 = 202.2 (2471-70-7) Content Minimum 95.0%, calculated by the normalisation
White or almost white, crystalline powder. procedure.
Melting point, 201 ° to 206°. Methyl Arachidate Methyl eicosanoate;
Methoxyphenylacetic Acid (RS)-2-Methoxy-2- CZIH420Z = 326.6 (1120-28-1)
phenylacetic acid; C 9H lO0 3 = 166.2 (702 1-09-2) Content, minimum 98.0%, determined by gas
White, crystalline powder or white or almost white crystals, chromatography (2.4.22).
sparingly soluble in water, freely soluble in ethanol (96%). White or yellow, crystalline mass, soluble in ethanol (96%)
Melting point, about 70°. and in light petroleum.
Methoxyphenylacetic Reagent Dissolve 2.7 g of Melting point, about 46°.
methoxyphenylacetic acid in 6 mL of tetramethylammonium MethyI Behenate See Methyl docosanoate.
hydroxide solutwn and add 20 mL of anhydrous ethanol. MethyI Benzenesulfonate Methyl benzenesulfonate;
Store in a polyethylene container. C 7H 8 0 3S = 172.2 (80-18-2)
3-MethoXY-L-tyrosine Clear, colourless liquido
C lOH 13 N0 4H 20 = 229 .2 (200630-46-2) Boiling point, about 148°.
Off-white or yellow powder. MethyI Benzoate Benzoic acid, methyl ester;
Methyl Acetate C 3H 60 2 = 74.1 (79-20-9) C 8 H gO z = 136.2 (93-58-3)
Clear, colourless liquid, soluble in water, miscible with Colourless liquid; d¡O, 1.088; boiling point, about 200°.
ethanol (96%) . 3-Methylbenzothiazolin-2-one hydrazone hydrochloride
d~g, about 0.933; ni?, about 1.361; boiling point, 56° to 58°. See Methylbenzothiazolone hydrazone hydrochloride.
MethyI4-acetylbenzoate C lOH lO 0 3 = 178.2 (3609-53-8) Methylbenzothiazolone hydrazone hydrochloride
Melting point, about 94°. 3-Methylbenzothiazol-2(3H)-one hydrazone hydrochloride
monohydrate; C 8 H lO CIN3S,H zO = 233.7 (38894-11-0)
MethyI 4-acetylbenzo~te Reagent Dissolve 0.25 g of
methyl 4-acetylbenzoate in a mixture of 5 mL of sulfun'c acid Almost white or yellowish, crystalline powder.
and 85 mL of cooled methanol. Melting point, about 270°.
MethylaI Dimethoxymethane, dioxapentane, formaldehyde Suitability for determination of aldehydes To 2 mL of
dimethyl acetal, methylene dimethyl ether; aldehyde-free methanol add 60 ~L of a 1 giL solution of
C 3 H g 0 2 = 76.1 (109-87-5) propionaldehyde in aldehyde-free methanol and 5 mLaf a 4 gIL
Clear, colourless, volatile, flammable liquid, soluble in water solution of methylbenzothiazolone hydrazonehydrochloride.
and miscible with ethanol (96%). Mix. Allow to stand for 30 minutes. Prepare ablank omitting
the propionaldehyde solution. Add 25.0 mL ofa 2 gIL
d~g, about 0.860; ni?, about 1.354; boiling point, about 41 °.
solution of femc chloride to the test solution andto the blank,
Methylal used in gas chromatography complies with the following dilute to 100.0 mL with acetone and mix. Theabsorbance
additional test. (2.2.25) of the test solution, measured at 660 nmusing the
Content, minimum 99.5%, determined by gas blank as compensation liquid, is not less than 0.62 .
chromatography. (R)-( + )-ex-MethylbenzyI Isocyanate (+ )-(R)-ex-
MethyI4-aminobenzoate C8H9NOZ = 151.2 (619-45-4) Methylbenzylisocyanate; (+)- [(1 R ) -1-
Melting point, 110° to 113°. Isocyanatoethyl]benzene; (+ )-(IR)-I-Phenylethyl isocyanate;
4-Methylaminophenol sulfate C 9 H 9NO = 147.2 (33375-06-3)
C14H2oNz06S = 344.4 (55-55-0) Content, minimum 99.0%; colourless liquid; d~g, about
Colourless crystals, very soluble in water, slighdy soluble in 1.045; ni?, about 1.513; boiling point, 55° to 56° at 2.5 mm
ethanol (96%). Hg.
Melting point, about 260°. Enantiomeric purity, minimum 99.5% .
MethylaminophenoI-Sulfite Reagent Store at a temperature of 2° to 8°.
Methylaminophenol-sulfite reagent (S)-( -)-ex-MethylbenzyI Isocyanate (- )-(S)-ex-
Dissolve 0.1 g of 4-methylaminophenol sulfate, 20 g of sodium Methylbenzyl isocyanate; (-)-[(1S)-I-
metabisuljite and 0.5 g of anhydrous sodium suljite in sufficient Isocyanatoethyl]benzene; (-)-(1S)-I-Phenylethyl isocyanate;
water to produce 100 mL. C 9 H 9NO = 147.2 (14649-03-7)
Methylamphetamine Hydrochloride Methamphetamine Content, minimum 99 .0%; colourless liquid; d~g, about
hydrochloride; C lOH 16CIN = 185.7 1.045; n~o about 1.514; boiling point 55° to 56° at 2.5 mm
Hg.
Enantiomeric purity, minimum 99.5%.
MethyI Anthranilate Methyl 2-aminobenzoate;
C8H9NOZ = 151.2 (134-20-3) Store at a temperature of 2° to 8°.
Colourless crystals or a colourless or yellowish liquid, soluble NOTE: do not use the reagent if it is coloured.
in water, freely soluble in ethanol (96%) . 2-Methylbutane Isopentane; CSH 1Z = 72.2 (78-78-4)
Boiling point, 134° to 136°; melting point, 24° to 25°. Content, minimum 99.5% of CSH 1Z'
Methyl anthranilate used in gas chromatography complies with the Very flarnmable colourless liquido
following additional test. d~g, about 0.621; ni? about 1.354; boiling point about 29°.
Assay Gas chromatography (2.2.28) as prescribed in the Water (2.5.12) Maximum 0.02%.
monograph Bitter-orange-fiower oil (1175) . Residue on evaporation Maximum 0.0003%.
Minimum transmittance (2.2.25) Using water as Fine, white or almost white powder, slightly soluble in water,
compensation liquid : 50% at 210 nm, 85% at 220 nm, 98% soluble in ethanol (96%).
at 240 nm and at higher wavelengths . Melting point, 300°, with decomposition.
2-Methylbut-2-ene CsH IO = 70.1 (513-35-9) 4,4 ' -Methylenebis-N,N-dimethylaniline
Very ftammable liquid, practically insoluble in water, miscible See Tetramethyldiaminodiphenylmethane
with ethanol (96%). 4,4'-Methylenebis-N,N-dimethylaniline Reagent See
Boiling point, 37S to 38S. Tetramethyldiaminodiphenylmethane reagent
Methyl Caprate See Methyl deeanoate. Methylene blue C152015,
Methyl Caproate Methyl hexanoate; 3,7-Dimethylaminophenothiazin-5-ium chloride;
C 7H 140 2 = 130.2 (106-70-7) Cl óH1 8CIN3S,xH20 = 319.9 (anhydrous) (122965-43-9)
dig, about 0.885; n5°, about 1.405; boiling point, 150° to lt occurs in different hydrated forms and may contain up to
151 °. 22% of water. A dark-green or bronze, crystalline powder,
Methyl Caprylate Methyl octano ate; freely soluble in water, soluble in ethanol (96 %) .
C 9 H 180 2 = 158.2 (111-11-5) Methylene chloride Dichloromethane;
d;g, about 0.876; n~, about 1.417; boiling point, 193 to
0 CH 2CI 2 = 84.9 (75-09-2)
194°. Colourless liquid, sparingly soluble in water, miscible with
Methylcellulose 450 (9004-67-5) ethanol (96%).
Methy1cellulose of the British Pharmacopoeia. Boiling point, 39° to 42°.
Methylene ehloride used in fluorimetry eomplies with the following
Nominal viscosity, 450 mPa·s.
additional test.
Methyl Cinnamate C IO H IO 0 2 = 162.2 (103-26-4)
Fluorescence Under irradiation at 365 nm, the
Colourless crystals practically insoluble in water, soluble in ftuorescence (2.2.21) measured at 460 nm in a 1 cm cell is
ethanol (96%).
not more intense than that of a solution containing
n5°, about 1.56; boiling point, about 260°; melting point, 34° 0.002 ppm of quinine in 0.5 M sulfurie acid measured in the
to 36°. same conditions.
Methyl Decanoate Methyl caprate; Methylene chloride, acidified
C ll H 22 0 2 = 186.3 (110-42-9)
To 100 mL of methylene ehloride add 10 mL of hydrochlorie
Content, minimum 99 .0%. acid, shake, allow to stand and separate the two layers.
Clear, colourless or yellow liquid, soluble in light petroleum. Use the lower layer.
n5°, 1.425 to 1.426; d;g, 0.871 to 0.876. Methyl Erucate Methyl cis-13-docosenoate;
Foreign substances Gas chromatography (2.2.28), C23H440 2 = 352.6 (1120-34-9)
injecting equal volumes of each of the following: d;g, about 0.871; n5°, about 1.456.
A 0.02 giL solution of the substance to be examined in 3-0-Methylestrone See 3-Methoxy-1,3,5 (1 0)-estratrien-17-
earbon disulfide (solution A), a 2 giL solution of the substance one
to be examined in earbon disulfide (solution B), and earbon Methyl Ethyl Ketone See Butan-2-one.
disulfide (solution C). Carry out the chromatographic
Methyleugenol 1,2-Dimethoxy-4-prop-2-enylbenzene;
pro ce dure under the conditions of the test for butylated C ll H 14 0 2 = 178.2 (93-15-2)
hydroxytoluene prescribed in the monograph Woolfat (0134).
The total area of any peaks, apart from the solvent peak and Methyleugenol used in gas chromatography complies with
the principal peak, in the chromatogram obtained with the following additional test.
solution B is less than the area of the principal peak in the Assay Gas chromatography (2.2.28) as prescribed in the
chromatogram obtained with solution A. monograph Niaouli oil, cineole type (2468).
Methyl Docosanoate Methyl behenate; Content Minimum 97.0%, ca1culated by the normalisation
C23H4ó0 2 = 354.6 (929-77-1) procedure.
Melting point, 54° to 55°. N-Methylglucamine Meglumine;
Methyldopa, racemic C IOH I3 N0 4 , 1Y2 H2 0 = 238.2 C 7H 17N O s = 195 .2 (6284-40-8)
Mixture of equal volumes of (2S)- and (2R) -2-amino-3-(3,4- General reagent grade of commerce.
0
dihydroxyphenyl)-2-methylpropanoic acids. Melting point, about 130 •
3-0-Methyldopamine hydrochloride Methyl Green CI 42585; basic blue 20, 4-[[4-(Dimethyl-

4-(2-Aminoethyl)-2-methoxyphenol hydrochloride; amino)phenyl] [4-(dimethyliminio)cyclohexa-2,5-
C 9 H 14CIN0 2 = 203.7 (1477-68-5) dienylidene]-methylphenyl] trimethylammoni um dichloride;
Melting point, 213° to 215 °. C2óH33CI2N3 = 458.5 (7114-03-6)
4-0-Methyldopamine hydrochloride Green powder, soluble in water, soluble in sulfuric acid
5-(2-Aminoethyl)-2-methoxyphenol hydrochloride; giving a yellow solution turning green on dilution with water.
C g H 14 CIN0 2 = 203.7 (645-33-0) Methyl Green-Iodomercurate Paper lmmerse thin
Melting point, 20r to 208°. strips of suitable filter paper in a 4% w/v solution of methyl
Methyl Eicosenoate Methyl cis-l 1-eicosenoate; green and allow to dry in airo lmmersethe strips for 1 hour in
C21H400 2 = 324.5 (2390-09-2) a solution containing 14% w/v of potassium iodide and
20% w/v of mercun'c iodide. Wash with distilled water until the
washings are practically colourless and allow to dry in airo
Methylenebisacrylamide
N,N'-Methylenebispropenamide; Store protected from light; use within 48 hours.
C 7HIQN 20 2 = 154.2 (110-26-9)
2014 Appendix I A V-AS7
Methyl4-Hydroxybenzoate See Methyl Methyl Margarate Methyl heptadecanoate;

parahydroxybenzoate CI sH 360 2 = 284.5 (1731-92-6)
l-Methylimidazole l-Methyl-lH-imidazole; White or almost white powder.
C 4H 6N 2 = 82.1 (616-47-7) Melting point, 32° to 34°.
Colourless or slightly yellowish liquid. Methyl margarate used in the assay of total fatty acids in Saw
n~o, about 1.495; boiling point, 195° to 197°. Palmetto Fruit complies with the following additional test.
Store in an airtight container, protected from light. Assay Examine by gas chromatography (2.2.28) as
l-Methylimidazole Rl prescribed in the monograph on Saw palmetto fruit.
Complies with the requirements prescribed for 1- The content is not less than 97%, calculated by the
methylimidazole with the following additional requirement. nOl7nalisation procedure.
Content, minimum 95.0%. Methyl Methacrylate Methyl 2-methylprop-2-enoate;
2-Methylimidazole C 4 H 6 N 2 = 82.1 (693-98-1) C SH S 0 2 = 100.1 (80-62-6)
White or almost white, crystalline powder. Colourless liquido
0
n~o, about 1.414; boiling point, about 100 ; melting point,
about - 48°.
MethylIodide Iodomethane; CH 31 = 141.9 (74-88-4)
It contains a suitable stabilising agent.
General reagent grade of commerce
Methyl Methanesulfonate C 2H 60 3S = 110.1 (66-27-3)
Methyl Isobutyl Ketone 4-Methyl-2-pentanone;
C 6H I20 = 100 .2 (108-10-1) Clear, colourless or slightly yellow liquido
Clear, colourless liquid, slightly soluble in water, miscible Content, minimum 99.0%; density, about 1.3 g cm-3 (25°).
n~, about 1.414; boiling point, about 202
0
with most organic solvents. •
d~g, about 0.80; boiling point, about 115°. Methyl N-methylanthranilate Methyl
Distillation range (2.2.11). Distil 100 mL. The range of 2-(methylamino)benzoate; C 9H 11 N0 2 = 165.2 (85-91-6)
temperature of distillation from 1 mL to 95 mL of distillate Pale yellow liquid; d¡O, about 1.128; n~o, about 1.579;
does not exceed 4.0°. boiling point, 255 10 258 0.
0
Residue on evaporation Maximum 0.01 % determined by Methyl N-methylanthranilate used in gas chromatography
evaporating on a water-bath and drying at 100-105°. complies with the following additional test.
Methyl Isobutyl Ketone Rl Assay Gas chromatography (2. 2.28), as prescribed in the
Shake 50 mL of freshly distilled methyl isobutyl ketone with monograph on Mandarin oil using the substance to be
0.5 mL of hydrochloric acid R1 for 1 mino Allow the phases 10 examined as the test solution.
separate and discard the lower phase. Prepare immediately Content, minimum 97%, calculated by normalisation.
before use. Methyl Myristate Methyl tetradecanoate;
Methyl Isobutyl Ketone R3 CIsH300 2 = 242.4 (124-10-7)
Complies with the requirements for methyl isobutyl ketone and Content, minimum 98.0%, determined by gas
with the following limits. chromatography (2.4.22).
Cr, maximum 0.02 ppm; Cu: maximum 0.02 ppm; Pb, Colourless or slightly yellow liquid, soluble in ethanol (96%)
maximum 0.1 ppm; Ni, maximum 0.02 ppm; Sn, maximum and in light petroleum.
0.1 ppm. di8, about 0.87; n~o, about 1.437; melting point, about 20°.
Methyl Laurate Methyl dodecanoate; 2-Methyl-1,4-naphthoquinone See M enadione.
C 13H 26 0 2 = 214.4 (111-82-0) Methyl Nervonate See Tetracos-15-enoic acid methyl estero
Content, minimum 98.0%, determined by gas 2-Methyl-5-nitroimidazole C 4H sN 3 0 2 = 127.1 (88054-
chromatography (2.4.22) . 22-2)
Colourless or yellow liquid, soluble in ethanol (96%) and in White 10 light yellow powder.
light petroleum. 0
Melting point, 252 to 254°.
d~g, about 0.87; n~o, about 1.431 ; melting point, about 50.
Content, minimum 98 .0%.
Methyl Lignocerate Methyl tetracosanoate; 2-Methyl-2-nitropropane-l,3-diol 2-Nitro-2-methyl-
C2sHsoOz = 382.7 (2442-49-1) 1,3-propanediol; C 4H g N0 4 = 135.1 (77-49-6)
Flakes. General reagent grade of commerce.
Methyl Linoleate Methyl (9Z,12Z)-octadeca-9,12- Methyl Oleate Methyl (Z)-octadec-9-enoate;
dienoate; CI 9H 340 2 = 294.5 (112-63-0) C1 9H 360 2 = 296.5 (112-62-9)
dig, about 0.888; n~, about 1.466; boiling point, 207° 10 Content, minimum 98.0%, determined by gas
208°. chromatography (2.4.22).
Methyl Linolenate Methyl (9Z, 12Z, 15Z)-octadeca- Colourless or slightly yellow liquid, soluble in ethanol (96%)
9,12,15-trienoate; C 1gH 32 0 2 = 292.5 (301-00-8) and in light petroleum.
dig, about 0.901; n~, about 1.471; boiling point, about d~g, about 0.88; n~o , about 1.452.
207°. Methyl0range CI 13025; Sodium
Methyl y-linolenate Methyl (6Z,9Z,12Z)-octadeca-6,9,12- 4 ' -dimethylaminoazo benzene-4-sulfona te;
trienoate; CI 9H 320 2 = 292.5 (16326-32-2) CI4H1 4N3Na03S = 327.3 (547-58-0)
Content, minimum 99.0% determined by gas chromatography. Orange-yeIlow, crystalline powder, slightly soluble in water,
practically insoluble in ethanol (96%).
Methy1 Orange Mixed Solution Dissolve 20 mg of Methy1phenyloxazoly1benzene 1,4-Bis [2-( 4-methyl-5-

methyl orange and 0.1 g of bromocresol green in 1 mL of phenyl)oxazolyl]benzene; CZ6H20N20 2 = 392.5 (3073-87-8)
0.2M sodium hydroxide and dilute to 100 mL with water. Fine, greenish-yellow powder with a blue fiuorescence or
Colour change pH 3.0 (orange) to 4.4 (olive-green). small crystals, soluble in ethanol (96%), sparingly soluble in
Methy1 Orange Solution xylene.
Dissolve 0.1 g of methyl orange in 80 mL of water and dilute Melting point, about 233°.
to 100 mL with ethanol (96%). Methylphenyloxazolylbenzene used lor liquid scint¡71ation is 01 a
Test for sensitivity A mixture of 0.1 mL of the methyl suitable analytical grade.
orange solution and 100 mL of carbon dioxide-free water is l-Methyl-4-phenyl-l ,2,3 ,6-tetrahydropyridine MPTP;
yellow. Not more than 0.1 mL of 1 M hydrochloric acid is C l2 H l5N = 173.3 (28289-54-5)
required to change the colour to red. White or almost white, crystalline powder, slighdy soluble in
Colour change pH 3.0 (red) to pH 4 .4 (yellow). water.
Methy1 Orange-Xy1ene Cyano1 FF Solution Dissolve Melting point, about 41 0 .
0.1 g of methyl orange and 0.26 g of xylene cyanol FF in Methylpiperazine 1-Methylpiperazine;
50 mL of ethanol (96%) and add sufficient water to produce C 5H l2N 2 = 100.2 (109-01-3)
100 mL. Colourless liquid, miscible with water and with ethanol
Methy1 Palmitate Methyl hexadecanoate; (96%).
C 17H3402 = 270.5 (112-39-0) d~g, about 0.90; ntO, about 1.466; boiling point, about 138°.
Content, minimum 98.0%, determined by gas
N-Methylpiperazine See Methylpiperazine
chromatography (2.4.22).
4-( 4-Methy1piperidin-l-y1)pyridine 4-
White or yellow, crystalline mass, soluble in ethanol (96%) (4-Methylpiperidino )pyridine;
and in light petroleum.
C11Hl6NZ = 176.3 (80965-30-6)
Clear liquido
Methy1 Palmito1eate Methyl cis-9-hexadecenoate;
n~, about 1.565.
C 17 H 32 0 2 = 268.4 (1120-25-8)
2-Methylpropanol Isobutyl alcohol, 2-Methylpropan-l-ol;
d~g, about 0.876; ntO, about 1.451.
C 4H IO O = 74.12 (78-83-1)
Methy1 Parahydroxybenzoate
Clear colourless liquid, soluble in water, miscible with
Of the British Pharmacopoeia. ethanol (96%).
Methy1 Pe1argonate Methyl nonanoate; d~g, about 0.80; ni], 1.397 to 1.399; boiling point, about
C IOH 200 2 = 172.3 (1731-84-6) 107°.
Clear, colourless liquido d~o, about 0.873; n~, about 1.422; Distillation range (2.2.11) Not les s than 96% distils
boiling point, 91 ° to 92°. between lOr and 109°.
Methyl pelargonate used in the assay 01 totallatty acids in Saw 2-Methylpropan-l-ol See 2-Methylpropanol.
Palmetto Fruit complies with the lollowing additional test. 2-Methylpropan-2-o1 See 2-Methyl-2-propanol.
Assay Examine by gas chromatography (2.2.28) as 2-Methyl-2-propano1 tert-Butyl alcohol, 1,I-Dimethyl
prescribed in the monograph on Saw palmeuo fruit. ethyl alcohol; C 4H IO O = 74.1 (75-65-0)
The content is not less than 98%, calculated by the
Clear, colourless liquid or crystalline mas s, soluble in water,
miscible with ethanol (96%).
2-Methy1pentane Isohexane; C 6H l4 = 86.2 (107-83-5)
Freezing point (2.2.18) About 25°.
Colourless, fiammable liquid, practically insoluble in water,
Distillation range (2.2. 11) Not les s than 95% distils
miscible with anhydrous ethano!.
between 81 ° and 83°.
d~g, about 0.653; boiling point, about 60.0°.
(15R)-15-Methy1prostaglandin F 2o: (5Z)-7-
3-Methy1pentan-2-one sec-Butyl methyl ketone; [(IR,2R,3R,5S)-3,5-Dihydroxy-2-[(IE)-(3R)-3-hydroxy-3-
C 6H l2 0 = 100.2 (565-61-7) methyloct-1-enyl] cyc1opentyl]hept-5-enoic
Colourless, fiammable liquido acid;C21H3605 = 368.5 (35864-81-4)
d~g, about 0.815; n~, about 1.400; boiling point, about Available as a 10 mg/mL solution in methyl acetate.
+118°. Store at a temperature below - 15°.
4-Methy1pentan-2-o1 4-Methyl-2-pentanol; N-Methylpyrrolidine C 5HIlN = 85 .2 (120-94-5)
C 6H l4 0 = 102.2 (108-11-2)
Clear, colourless, volatile liquido
d~o, about 0.802; n~, about 1.411; boiling point, about
N-Methy1pyrrolidone l-Methylpyrrolidin-2-one;
132°.
C 5H 9NO = 99 .1 (872-50-4)
4-Methy1pentan-2-one See Methyl isobutyl ketone.
d~g, about 1.028; boiling point, about 202°; melting point,
4-Methy1pentan-2-one Rl See Methyl isobutyl ketone R1 . about - 24°.
4-Methy1pentan-2-one R3 See Methyl isobutyl ketone R3. Methyl Red Cl 13020; 2-
4-Methy1phenazone 1,5-Dimethyl-2-(4-methylphenyl)- (4-dimethylaminophenylazo )benzoic acid;
1,2-dihydro-3H-pyrazol-3-one; Cl5Hl5N302 = 269.3 (493-52-7)
Cl 2Hl4N20 = 202.3 (56430-08-1) Dark-red powder or violet crystals, practically insoluble in
Methyl Red Mixed Solution Dissolve 0.1 g of methyl red Molybdenum(vr) Oxide Molybdenum trioxide;
and 50 mg of methylene blue in 100 mL of ethanol (96%). Mo0 3 = 143.9 (1313-27-5)
Colour change pH 5.2 (red-violet) ro pH 5.6 (green). Analytical reagent grade of commerce.
Methyl Red Solution Molybdovanadic Reagent In a 150 mL beaker, mix 4 g
Dissolve 50 mg in a mixture of 1.86 mL of 0. 1 M sodium of finely powdered ammonium molybdate and 0.1 g of finely
hydroxide and 50 mL of ethanol (96%)and dilute to 100 mL powdered ammonium vanadate. Add 70mL of zvater and
with water. grind the partic!es using a glass rod. A c1ear solution is
Testfor sensitivity To 0.1 mL ofthe methyl red solution obtained within a few minutes. Add 20 mL of nitric acid and
dilute ro 100 mL with water.
add 100 mL of carbon dioxide-free water and 0.05 mL of
0.02 M hydrochloric acid. The solution is red. Not more than Monodocosahexaenoin Monoglyceride of
0.1 mL of 0.02 M sodium hydroxide is required to change the docosahexaenoic acid (C22:6); Glycerol
colour ro yellow. monodocosahexaenoate; (all-Z)-Docosa-4, 7,10,13,16,19-
Colour change pH 4.4 (red) to pH 6.0 (yellow) . hexaenoic acid, mono es ter with propane-l,2,3-triol;
CZSH3S04 = 402.6 (124516-13-8)
Methyl Salicylate C SH S 0 3 = 152.2 (119-36-8)
The reagent from Nu-Chek Prep, Inc. has been found
Of the British Pharmacopoeia. suitable.
When used in the Assay of Methyl Salicylate preparations, Mordant Black 11 CI 14645; eriochrome black T;
use a grade containing not less than 99 .0% of C SH S 0 3 . solochrome black; C2oHIZN3Na07S = 461.4 (1787-61-7)
Methyl Stearate Methyl octadecanoate; Brownish-black powder, soluble in water and in ethanol
ClgH3S0Z = 298.5 (112-61-8) (96%) .
Content, minimum 98 .0%, determined by gas Srore in an airtight container, protected from light.
chromarography (2.4.22).
Mordant Black 11 Mixed Triturate A mixture of 1 g of
White or yellow, crystalline mass, soluble in ethanol (96%) mordant black 11, 0.4 g of methyl orange and 100 g of sodium
and in light petroleum. chloride.
Melting point, about 38°. Complies with the following test.
5-Methylthiazol-2-ylamine See 2-Amino-5-methylthiazole. Sensitivity to magnesium Dissolve 50 mg in 100 mL of
Methyl Thymol Blue See Methylthymol blue. water; a brown colour is produced. Add 0.3 mL of
Methylthymol Blue Tetrasodium 2,2 ' ,2",2 /1f -[3H-2,1- 6M ammonia; the colour changes to pale green. Add 0.1 mL
benzoxathiol-3-ylidenebis [[ 6-hydroxy-2-methyl-5- of a 1.0% w/v solution of magnesium sulfate; the colour
(l-methylethyl)-3, l-phenylene ]methylenenitrilo]] tetraacetate changes ro red.
S,S-dioxide; C37H40NzNa4013S = 845 (1945-77-3) Mordant Black 11 Solution A 0.1 % w/v solution of
Produces a blue colour with calcium in alkaline solution. mordant black 11 in ethanol (96%).
Methylthymol Blue Mixture A mixture of 1 part of Mordant Black 11 Triturate
methylthymol blue and 100 parts of potassium nitrate. Mix 1 g of mordant black 11 with 99 g of sodium chloride.
N-Methyl-m-toluidine N,3-Dimethylaniline; N,3-
Testfor sensitivity Dissolve 50 mg in 100 mL of water.
Dimethylbenzenamine; Methyl-m-rolylamine;
The solution is brownish-violet. On addition of 0.3 mL of
C sH l1 N = 121.2 (696-44-6)
dilute ammonia R1 the solution tums blue. On the
Content, minimum 97%. subsequent addition of 0.1 mL of a 10 giL solution of
Methyl Tricosanoate Tricosanoic acid methyl ester; magnesium sulfate, it tums violet.
C24H4S02 = 368.6 (2433-97-8) Store in an airtight container, protected from light.
Content, minimum 99.0% .
Mordant black 11 triturate Rl
White or almost white crystals, practically insoluble in water,
soluble in hexane. Mix 1.0 g of mordant black 11, 0.4 g of methyl orange and
100 g of sodium chloride.
Melting point, 55° ro 56°.
Methyl Tridecanoate C14HzsOz = 228.4 (1731-88-0) Mordant Blue 3 Cl 43820; chromoxane cyanine
(3564-18-9)
Colourless or slightly yellow liquid, soluble in ethanol (96%)
and in light petroleum. General reagent grade of commerce.
d~g, about 0.86; nbo, about 1.441; melting point, about 6°. A dark red or reddish brown powder.
Methyl 3,4,5-Trimethoxybenzoate Produces an intense purple colour with aluminium in weakly
CllH 140 S = 226.23 (1916-07-0) acidic solutions . When metal ions are absent, for example, in
N-Methyltrimethylsilyl-trifluoroacetamide 2,2,2- the presence of an excess of disodium edetate, the solution is
Trifiuoro-N-methyl-N-(trimethylsilyl)acetamide; pale pink.
C 6 H 12F 3 NOSi = 199.3 (24589-78-4) Morphine, Anhydrous Add 5M ammonia, in slight excess,
nbo, about 1.380; boiling point, 130° ro 132°. to a solution of morphine sulfate in water, wash the
Minocycline Hydrochloride (13614-98-7) Of the British precipitated morphine with water until free from ammonium
Pharmacopoeia. salts and dry at 110°.
Molecular Sieve Molecular sieve composed of sodium Morphine Hydrochloride Of the British Pharmacopoeia.
aluminosilicate. It is available as beads with a pore size of Morpholine Tetrahydro-I,4-oxazine;
0.4 nm and with a diameter of 2 mm. C 4HgNO = 87.12 (110-91-8)
Molecular Sieve for Chromatography Molecular sieve Colourless, hygroscopic liquid, fiammable, soluble in water
composed of sodium aluminosilicate. The pore size is and in ethanol (96%) .
indicated after the name of the reagent in me tests where it d~g, about 1.01.
is used. If necessary, the partic!e size is also indicated.
Distillation range (2.2.11) Not les s than 95 % distils Myrtillin Delphinidin 3-0-glucoside chloride;
between 126° and 130°. CZIHzICIOlZ = 500.8 (6906-38-3)
Store in an airtight container. Nalorphine HydrochIoride CI9HzI N03,
Morpholine for Chromatography HCI = 347.8 (57-29-4)
Complies with the requirements prescribed for morpholine General reagent grade of commerce.
with the following additional requirement. Naphthalene CIOHs = 128.2 (91-20-3)
Content, minimum 99.5% . White or almost white crystals, practically insoluble in water,
Murexide 5,5 ' -Nitrilobis(pyrimidine-2,4,6(l H,3H,5H)- soluble in ethanol (96%).
trione) monoammonium saltó CsHsN 60 6,HzO = 302.2 Melting point, about 80°.
Brownish-red crystalline powder, sparingly soluble in cold N aphthalene used for liquid scintillation is of a suitable analytical
water, soluble in hot water, practically insoluble in ethanol grade.
(96%), soluble in solutions of potassium hydroxide or Naphthalene-l,3-dio1 1,3-Dihydroxynaphthalene;
sodium hydroxide giving a blue colour. naphthoresorcinol; CIOHsOz = 160.2 (132-86-5)
Myosmine 3-(4,5-Dihydro-3H-pyrrol-2-yl)pyridine; Crystalline, generally brownish-violet powder, freely soluble
C 9HIONz = 146.2 (532-12-7) in water and in ethanol (96%).
Colourless crystals. Melting point, about 125°.
Melting point, about 45°. Naphthalene-2, 7-diol 2,7 -Dihydroxynaphthalene;
~-Myrcene 7 -Methyl-3-methylenocta-l,6-diene; CIOHsOz = 160.2 (582-17-2)
CIOHI6 = 136.2 (123-35-3) Needles, soluble in water and in ethanol (96%).
Oily liquid with a pleasant odour, practically insoluble in Melting point, about 190°.
water, miscible with ethanol (96%), soluble in glacial acetic Naphthalenediol Reagent Solution Dissolve 2.5 mg of
acid. It dissolves in solutions of alkali hydroxides . naphthalene-2,7-diol in 90 mL of methanol and add 10 mg of
d¡O, about 0.794; nf,O, about l.470. potassium hexacyanoferrate(m) and 50 mg of potassium cyanide
fJ-Myrcene used in gas chromatography complies with the dissolved in 10 mL of water. AlIow to stand for 30 minutes
following additional test. and add 100 mL of 0.05M sodium hydroxide.
Assay Gas chromatography (2.2.28) as prescribed in the Naphthalenediol Solution 2,7-Dihydroxynaphthalene
monograph Peppermint oil (0405). solution.
Test solution The substance to be examined. Dissolve 10 mg of 2,7-dihydroxynaphthalene in 100 mL of
Content Minimum 90.0%, calculated by the normalisation sulfun'c acid and allow to stand until decolorised.
procedure. U se within 2 days.
Myristic Acid Tetradecanoic acid; Naphtharson Thorin; disodium 4-[(2-arsonophenyl)azo]-
C1 4HzsOz = 228.4 (544-63-8) 3-hydroxynaphthalene-2,7 -disulfonate;
Colourless or white or almost white fiakes . CI 6HIIAsNzNazOlOSZ = 576 .3 (3688-92-4)
Melting point, about 55°. Red powder, soluble in water.
Myristic acid used in the assay of total fatty acids in Saw Naphtharson Solution A 0.058% w/v solution.
Palmetto Fruit complies with the following additional test. Sensitivity To 50 mL of ethanol (96%), add 20 mL of
Assay Examine by gas chromatography (2.2.28) as water, 1 mL of 0.05M sulfuric acid and 1 mL of the
prescribed in the monograph on Saw palmetto fruit. naphtharson solution. Titrate with 0.025M barium perchlorate
The content of myristic acid is not les s than 97 %, calculated
VS; the colour changes from orange-yellow to orange-pink.
by the normalisation procedure. Store protected from light; use within one week.
Myristicine 5-AlIyl-1-methoxy-2,3- IX-Naphthol See l-Naphthol.
methylenedioxybenzene, 4-Methoxy-6-(prop-2-enyl)-1,3- IX-Naphthol Solution See l-Naphthol solution.
benzodioxole; C ll H 12 0 3 = 192.2 (607-91-0) ~-Naphthol See 2-Naphthol.
Oily colourless liquid, practically insoluble in water, slightly ~-Naphthol Solution See 2-Naphthol solution.
soluble in anhydrous ethanol, miscible with toluene and with
~-Naphthol Solution Rl Dissolve 3.0 mg of 2-naphthol
xylene.
inin 50 mL of sulfuric acidand dilute to 100.0 mL with the
d~g, about l.l44; nf,O, about l.540; boiling point, 276° to same acid. Use the recentlyprepared solution.
277°; melting point, about 173°.
l-Naphtho1 IX-Naphthol; CIOHsO = 144.2 (90-15-3)
Chromatography. Thin-Iayer chromatography (2.2.27) as
prescribed in the monograph Star anise (1153); the White or almost white, crystalline powder or colourless or
chromatogram shows only one principal spot. white or almost white crystals, darkening on exposure to
light, slightly soluble in water, freely soluble in ethanol
Myristicine used in gas chromatography complies with the (96%).
Assay. Gas chromatography (2.2.28) as prescribed in the
monograph Nutmeg oil (1552). Store protected from light.
Content Minimum 95.0%, calculated by the normalisation 2-Naphthol ~-Naphthol; CIOHsO = 144.2 (135-19-3)
procedure. White or slightly pink plates or crystals, very slightly soluble
Store protected from light. in water, very soluble in ethanol (96%).
Myristy1 Alcohol Tetradecan-1-01; Melting point, about 122°.
CJ4H 30 0 = 214.4 (112-72-1) Store protected from light.
d~g, about 0.823; melting point, 38° to 40°.
l-Naphthol Solution Dissolve 0.10 g of 1-naphthol in Naringin 7 -[[2-0-(6-Deoxy-a-L-mannopyrano syl) -~-D

3 rnL of a 15% w/v solution of sodium hydroxide and dilute glucopyranosyl] oxy]-5-hydroxy-2-(4-hydroxyphenyl)-2,3-
to 100 rnL with water. Prepare irnmediately before use. dihydro-4H-chromen-4-one;
l-Naphthol Solution, Strong Dissolve 1 g of 1-naphthol CZ7H3201 4 = 580.5 (10236-47-2)
in a solution of 6 g of sodium hydroxide and 16 g of anhydrous White or almost white crystalline powder, slightly soluble in
sodium carbonate in 100 rnL of water. water, soluble in methanol and in dimethylformamide.
2-Naphthol Solution Dissolve 5 g of freshly recrystallised Melting point, about 171 °.
2-naphthol in 40 rnLof dilute sodium hydroxide solution and Absorbance Naringin dissolved in a 0.5 % w/v solution of
dilute to 100 rnLwith water. Prepare irnrnediately before use. dimethylformamide in methanol shows an absorption maximum
Naphtholbenzein See 1-Naphtholbenzein. at 283 nm.
Naphtholbenzein Solution See 1-Naphtholbenzein solution. Neohesperidin Hesperetin-7 -neohesperidoside, (2S)-7 -[ [2-
l-Naphtholbenzein Naphtholbenzein, O-(6-DeoXY-(x-L-mannopyranosyl)-~-D-glucopyranosyl]oxy]-5-
phenylbis(4-hydroxynaphthyl)rnethanol; hydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-4H-l-
C 27 H 1SO Z = 374.4 (145-50-6) benzopyran-4-one; C ZSH 34015 = 610 .6 (13241-33-3)
Brownish-red powder or shiny brownish-black crystals, trans-Nerolidol 3,7,11-Trimethyldodeca-l ,6, 10-trien-3-ol;
practically insoluble in water, soluble in ethanol (96%) and C l5 H zó O = 222.4 (40716-66-3)
in glacial acetic acid. Slightly yellow liquid, slight odour of lily and lily of the
l-Naphtholbenzein Solution A 0.2% w/v solution of 1- valley, practically insoluble in water and in glycerol, miscible
naphtholbenzein in anhydrous acetic acid. with ethanol (96%).
Sensitivity To 50 rnL of glacial acetic acid add 0.25 rnL of di8, about 0.876; n~o, about 1.479; boiling point, 145° to
the naphtholbenzein solution. The solution is brownish- 146°.
yellow. Not more than 0.05 mL of 0. 1 M perchloric acid is trans-Nerolidol used in gas chromatography complies with the
required to change the colour to green. following additional test.
Naphthol Yellow 2,4-Dinitro-l-naphthol, sodium salt; Assay Gas chromatography (2.2.28) as prescribed in the
CIOH5N zNa05 = 256.2 monograph Bitter-orange-fiower oi! (J 175).
Orange-yellow powder or crystals, freely soluble in water, Test solution The substance to be examined.
slightly soluble in ethanol (96%). Content Minimum 90.0%, ca1culated by the normalisation
Naphthol Yellow S 8-Hydroxy-5,7-dinitro-2- procedure.
naphthalenesulfonic acid disodium salto Disodium Neryl Acetate (Z)-3,7-Dimethylocta-2,6-dienyl acetate;
5,7-dinitro-8-oxidonaphthalene-2-sulfonate. Colour Index C12HzoOZ = 196.3 (141-12-8)
No. 10316; C IOH 4N zNa zOgS = 358.2 (846-70-8) Colourless, oily liquido
Yellow or orange-yellow powder, freely soluble in water.
di8, about 0.907; n~, about 1.460.
l-Naphthylacetic Acid (Naphthalen-l-yl)acetic acid; Neryl acetate used in gas chromatography complies with the
C12HIOOZ = 186.2 (86-87-3) following additional test.
White to yellow crystalline powder, very slightly soluble in Assay Gas chromatography (2.2.28) as prescribed in the
water, freely soluble in acetone. Melting point, about 135°. monogtaph Bitter-orange-fiower oil (J 175) .
2-Naphthylacetic Acid 2-Naphthaleneacetic acid; Test solution The substance to be examined.
C1zHIOO Z = 186.2 (581-96-4)
Content Minimum 93.0%, ca1culated by the normalisation
General reagent grade of commerce. procedure.
Light yellow to light brown crystals; melting point, about Neutral Red CI 50040; basic red 5;
142°. C 1sH 17 CIN4 = 288 .8 (553-24-2)
Naphthylamine See 1-Naphthylamine. Produces a red colour with acids and an orange colour with
l-Naphthylamine Naphthylamine, 1-aminonaphthalene; alkalis.
CIOH9N = 143.2 (134-32-7) Neutral Red Solution A 0.1 % w/v solution of neutral red
White or almost white, crystalline powder, turning pink on in ethanol (50%) (pH range, 6.8 to 8.0) .
exposure to light and air, slightly soluble in water, freely Nickel-Aluminium Alloy Raney nickel catalyst
soluble in ethanol (96%) .
Contains 48% to 52% of aluminium (Al; Ar 26.98) and
48% to 52% ofnickel (Ni; Ar 58.70).
Store protected from light. Before use, reduce to a fine powder (I80) (2.9.12).
Naphthylethylenediamine Dihydrochloride See N- It is practically insoluble in water and soluble in mineral
(J -Naphthyl) ethylenediamine dihydrochloride.
acids.
N-(l-Naphthyl)ethylenediamine Dihydrochloride N- Nickel-Aluminium Alloy (Halogen-free)
(I-Naphthyl)ethane-diamine dihydrochloride;
Contains 48% to 52% of alumnium (Al; Ar 26.98) and 48%
Cl zHl~Z,2HCI = 259.2 (1465-25-4)
to 52% ofnickel (Ni; Ar 58.71).
It may contain methanol of crystallisation.
Fine, grey powder, practically insoluble in water, soluble in
White or yellowish-white powder, soluble in water, slightly mineral acids with formation of salts.
soluble in ethanol (96%).
Chlorides Maximum 10 ppm.
Naphthylethylenediamine Dihydrochloride Solution
Dissolve 0.400 g in 40 mL of a mixture of 67 volumes of
Dissolve 0.1 g of naphthylethylenediamine dihydrochloride in sulfuric acid and 33 volumes of dilute nitric acid. Evaporate
water and dilute to 100 mL with the same solvent. Prepare the solution nearly to dryness, dissolve the residue in water
immediately before use.
and dilute to 20.0 mL with the same solvento To one half- 30 minutes, then filter and store at 2° to 8°. Irnmediately
aliquot of the solution, add 1.0 mL of 0.1 M silver nitrate. before use dilute 2.5 mL of the solution with 5 mL of water
Filter after 15 minutes and add 0.2 mL of sodium chloride and 45 mL of 2-propano!'
solution (containing 10 Ilg of chlorides per millilitre) to the Ninhydrin and Stannous Chloride Reagent Rl
filtrate. After 5 minutes the solution is more opalescent than Dissolve 4 g of ninhydrin in 100 mL of ethylene glycol
a mixture of the second half-aliquot of the solution with monomethyl ether. Shake gently with 1 g of cation exchange
1.0 mL of 0.1 M silver nitrate. resin (300 11m to 840 11m) and filter (solution A) . Dissolve
Nickel Chloride See Nickel(ll) chloride. 0.16 g of stannous chloride in 100 mL of buffersolution pH 5.5
Nickel(n) Chloride Anhydrous nickel chloride, nickel (solution B). Immediately before use, mixequal volumes of
chloride; NiCl 2 = 129.6 (7718-54-9) each solution.
Yellow, crystalline powder, very soluble in water, soluble in Ninhydrin Reagent 1 Transfer 10 litres of 2-
ethanol (96%). It sublimes in the absence of air and readily methoxyethanol to a clean, dry 20-litre bottle and purge with
absorbs arnmonia. The aqueous solution is acid. oxygen-free nitrogen for 2 to 3 minutes. Continue the nitrogen
Nickel(n) Chloride Hexahydrate fiow and add 10 g of hydrindantin and 100 g of ninhydrin
NiCI 2,6H 20 = 237 .7 (7791-20-0) and allow to dissolve; the solution will be straw coloured
when dissolution is complete. Add 2 litres of a solution
prepared by dissolving 5.444 kg of sodium acetate in 5 litres
Nickel Nitrate Hexahydrate Ni(N0 3)2,6H 2 0 of water, adding 1 litre of glacial acetic acid, adjusting the pH
= 290.8 (13478-00-7)
to 5.5 with glacial acetic acid and adding sufficient water to
Nickel Sulfate See Nickel(ll) sulfate. produce 10 litres. Add 6.5 litres of water and 20 mL of a
Nickel(n) Sulfate Nickel(n) sulphate, nickel sulfate, nickel 20% w/v solution of polyoxyethy lene 23 lauryl ether.
sulfate heptahydrate; NiS0 4 ,7H 20 = 280.9 (10101-98-1) The deep red reagent is stable for at least 8 weeks when
Green, crystalline powder or crystals, freely soluble in water, stored under nitro gen and protected from light; avoid
slightly soluble in ethanol (96%). exposure to arnmonia .
Nicotinamide-adenine Dinuc1eotide NAD+; Ninhydrin Solution A 0.2% w/v solution of ninhydrin in a
C21 H 27N7014P2 = 663 (7298-93-3) mixture of 5 volumes of dilute acetic acid and 95 volumes of
White or almost white powder, very hygroscopic, freely butano!.
soluble in water. Ninhydrin Solution Rl Dissolve 1.0 g of ninhydn·n in
Nicotinamide-adenine Dinuc1eotide Solution Dissolve 50 mL of ethanol (96%) and add 10 mL of glacial acetic acid.
40 mg of nicotinamide-adenine dinucleotide in water and dilute Ninhydrin Solution R2 Dissolve 3 g of ninhydrin in
to 10 mL with the same solvent. Prepare irnmediately before 100 mL of a 4.55% w/v solution of sodium metabisulfite.
use . Ninhydrin Solution R3 A 0.4% w/v solution of ninhydrin
Nicotinic Acid C ó H 5N0 2 = 123 .1 (59-67-6) in a mixture of 5 volumes of anhydrous acetic acid and
Of the British Pharmacopoeia. 95 volumes of butanol.
Nile Blue A CI 51180; 5-amino-9- Ninhydrin Solution R4 A 3 gIL solution of ninhydrin in a
(diethylamino )benzo [a]phenoxazinylium hydrogen sulfate; mixture of 5 volumes of glacial acetic acid and 95 volumes of
C 2oH 21N30 5S = 415.5 (3625-57-8) 2-propanol.
Green, crystalline powder with a bronze lustre, sparingly Nitrazepam Of the British Pharmacopoeia.
soluble in ethanol (96%), in glacial acetic acid and in Nitric Acid HN0 3 = 63.0 (7697-37-2)
pyridine. Content, 63 .0% m/m to 70.0% mimo
Absorbance (2.2.25). A 0.005 gIL solution in ethanol Clear, colourless or almost colourless liquid, miscible with
(50% v/v) shows an absorption maximum at 640 nm. water.
Nile B1ue A Solution dig, 1.384 to 1.416 .
A 10 giL solution in anhydrous acetic acid. A 10 gIL solution is strongly acid and gives the reaction of
Test/or sensitivity To 50 mL of anhydrous acetic acid add nitrates (2.3.1) .
0.25 mL of the Nile blue A solution. The solution is blue. Appearance Nitric acid is clear (2.2.1) and not more
On the addition of 0.1 mL of 0.1 M perchloric acid, the intensely coloured than reference solution Y ó (2.2.2,
colour changes to blue-green. Methad lI) .
Colour change pH 9.0 (blue) to pH 13.0 (red). Chlorides (2.4.4) Maximum 0.5 ppm .
Aqueous Nile Blue A Solution Dissolve 40 mg of Nile To 5 g add 10 mL of water and 0.3 mL of silver nitrate
Blue A in 200 mL of water, shake with 100 mL of n-heptane solution R2 and allow to stand for 2 minutes protected from
in a 500 mL separating funnel and discard the heptane layer. light. Any opalescence is not more intense than that of a
Repeat the extraction with four 100 mL quantities of standard prepared in the same manner using 13 mL of water,
n-heptane and mix 20 mL of the aqueous solution with 0.5 mL of nitric acid, 0.5 mL of ehloride standard solution
180 mL of ethanol. (5 ppm el) and 0.3 mL of silver nitrate solution R2.
Ninhydrin Indane-1 ,2,3-trione, 1,2,3-indanetrione Sulfates (2.4.13) Maximum 2 ppm.
monohydrate; C 9 H 40 3,H 20 = 178.1 (485-47-2) Evaporate 10 g to dryness with 0.2 g of sodium carbonate.
White or very pale yellow, crystalline powder, soluble in Dissolve the residue in 15 mL of distilled water. Prepare the
water and in ethanol (96 %). standard using a mixture of 2 mL of sulfate standard solution
Store protected from light. (10 ppm SO,J and 13 mL of distilled water.
Ninhydrin and Stannous Chloride Reagent Dissolve Arsenic (2.4.2, M ethod A ) Maximum 0.02 ppm.
0.2 g of ninhydrin in 4 mL of hot water, add 5 mL of a Gently heat 50 g with 0.5 mL of sulfuric acid until white
0.16% w/v solution of stannous chloride, allow to stand for fumes begin to evolve. To the residue add 1 mL of a 100 gIL
solution of hydroxylamine hydrochloride and dilute ro 2 mL Nitric Acid, Lead-free, Dilute

with water. Prepare the standard using 1.0 mL of arsenic Dilute 5 g of lead-free nitric acid R1 ro 100 mL with deionised
standard solution (1 ppm As). distilled water.
[ron (2.4.9) Maximum 1 ppm. Nitric Acid, Lead-free
Dissolve the residue from the determination of sulfated ash in Complies with the requirements prescribed for nitn'c acid with
1 mL of dilute hydrochloric acid and dilute to 50 mL with the foIlowing additional test.
water. Dilute 5 mL of this solution to 10 mL with water. Lead Maximum 0.1 ppm.
Heavy metals (2.4.8) Maximum 2 ppm. Atomic absorption spectrometry (2.2.23, Method IJ) .
Dilute 10 mL of the solution prepared for the limit test for
Test solution To 100 g add 0.1 g of anhydrous sodium
iron to 20 mL with water. 12 mL of the solution complies carbonate and evaporate to dryness. Dissolve the residue in
with test A. Prepare the reference solution using lead standard
water, heating slightly, and dilute ro 50.0 mL with the same
solution (2 ppm Pb). solvent.
Sulfated ash Maximum 0.001 %. Source: lead hoIlow-cathode lampo
CarefuIly evaporate 100 g to dryness. Moisten the residue
Wavelength: 283.3 nm or 217.0 nm.
with a few drops of sulfuric acid and heat to duIl red.
Atomisation device: air-acetylene flameo
Assay To 1.50 g add about 50 mL of water and ti trate
with 1 M sodium hydroxide, using 0.1 mL of methyl red Nitric Acid, Lead-free Rl
solution as indicator. Nitric acid containing not more than 1 ¡.tg/kg of lead.
1mL of 1 M sodium hydroxide is equivalent to 63.0 mg of Nitrilotriacetic Acid C 6 H 9 N0 6 = 191.1 (139-13-9)
HN0 3 · White or almost white crystaIline powder, practicaIly
Store protected from light. insoluble in water and in most organic solvents.
When no molarity is indicated use analytical reagent grade of Melting point, about 246°, with decomposition.
commerce containing about 70% w/w of HN0 3 . Nitroaniline See 4-Nitroaniline.
When solutions of molarity XM are required, they should be 4-Nitroaniline Nitroaniline;
prepared by diluting 63x mL of nitric acid to 1000 mL with C 6 H 6N 2 0 2 = 138.1 (100-01-6)
water. Bright yeIlow, crystaIline powder, very slightly soluble in
Nitric Acid, Cadmium- and Lead-free water, sparingly soluble in boiling water, soluble in ethanol
Complies with the requirements prescribed for nitric acid (96%), forms water-soluble saIts with strong mineral acids.
and with the foIlowing additional test. Melting point, about 148' .
Test solution To 100 g add 0.1 g of anhydrous sodium Nitroaniline Solution, Diazotised Dissolve 0.4 g of 4-
carbonate and evaporate ro dryness. Dissolve the residue in nitroaniline in 60 mL of 1M hydrochloric acid with the aid of
water heating slightly, and dilute to 50.0 mL with the same heat, cool to 15° and add a 10% w/v solution of sodium nitrite
solvent. until one drop of the mixture tums starch iodide paper blue.
Cadmium Maximum 0.1 ppm. Prepare immediately before use.
Atomic absorption spectrometry (2.2.23, Method IJ) . Nitrobenzaldehyde See 2-Nitrobenzaldehyde.
Source: cadmium hoIlow-cathode lamp. 2-Nitrobenzaldehyde Nitrobenzaldehyde;
Wavelength: 228.8 nm. C 7 H sN0 3 = 151.1 (552-89-6)
Atomisation device: air-acetylene or air-propane flame o YeIlow needles, slightly soluble in water, freely soluble in
Lead Maximum 0.1 ppm. ethanol (96%), volatile in steam.
Aromic absorption spectrometry (2.2.23, Method IJ).
Nitrobenzaldehyde Paper
Source: lead hoIlow-cathode lamp.
Wavelength: 283.3 nm or 217.0 nm. Dissolve 0.2 g ofnitrobenzaldehyde in 10 mL ofa 200 gIL
solution of sodium hydroxide. Use the solution within 1 hour.
Atomisation device: air-acetylene flameo Irnmerse the lower half of a slow filter paper strip 10 cm long
Nitric Acid, Dilute and 0.8-1 cm wide. Absorb the excess reagent between two
Contains about 125 gIL of HN0 3 (M, 63.0). sheets of filter paper. Use within a few minutes of
Dilute 20 g of nitric acid to 100 mL with water. preparation.
Nitric acid, Dilute Rl Nitrobenzaldehyde Solution
Dilute 40 g of nitric acid to 100 mL with water. Add 0.12 g of powdered nitrobenzaldehyde ro 10 mL of dilute
Nitric acid, Dilute R2 sodium hydroxide solution; aIlow to stand for 10 minutes
shaking frequently and filter. Prepare immediately before use.
Dilute 30 g of nitric acid ro 100 mL with water.
Nitrobenzene C 6 H sN0 2 = 123.1 (98-95-3)
Nitric Acid, Fuming HN0 3 = 63 .0 1 (52583-42-3)
ColourIess or very slightly yeIlow liquid, practicaIly insoluble
Clear, slightly yeIlowish liquid, fuming on contact with airo in water, miscible with ethanol (96%).
dig, about 1.5. Boiling point, about 211 o .
Nitric Acid, Heavy Metal-free Complies with the Dinitrobenzene To 0.1 mL add 5 mL of acetone, 5 mL of
requirements prescribed for nitric acid and with the foIlowing water and 5 mL of strong sodiu111 hydroxide solution. Shake and
maxirnum contents of heavy metals: aIlow to stand. The upper layer is almost colourless.
As: 0.005 ppm; Cd: 0.005 ppm; Cu: 0.001 ppm; Fe: 4-Nitrobenzoic Acid C 7 H sN0 4 = 167.1 (62-23-7)
0.02 ppm; Hg: 0.002 ppm; Ni: 0.005 ppm; Pb: 0.001 ppm;
YeIlow crystals; meIting point, about 240 ".
Zn: 0.01 ppm.
4-Nitrobenzyl Bromide C7H6BrNOz = 216.0 (100-11-8) Nitro-molybdovanadic Reagent See Nitro-vanado-

General reagent grade of commerce. molybdic reagent.
Pale yellow crystals with a lachrymatory vapour; melting 4-NitrophenoI p-Nitrophenol;
point, about 99 0
•
C 6H sN0 3 = 139 .1 (100-02-7)
Nitrobenzoyl Chloride 4-Nitrobenzoyl chloride; Content, minimum 95%.
C 7H 4 CIN0 3 = 185.6 (122-04-3) Colourless or slightly yellow powder, sparingly soluble in
Yellow crystals or a crystalline mas s, decomposing in moist water and in methano!. Melting point, about 114°.
air, completely soluble in sodium hydroxide solution giving a 3-Nitrosalicylic Acid 2-Hydroxy-3-nitrobenzoic acid;
yellowish-orange colour. C 7 H 5 N0 5 = 183.1 (85-38-1)
0
Melting point, about 73 • Yellowish crystals, slightly soluble in water, freely soluble in
Nitrobenzyl Chloride See 4-Nitrobenzyl chloride. ethanol (96%).
0
4-Nitrobenzyl Chloride Nitrobenzyl chloride; Melting point, 142 to 147°.
C 7 H 6CIN0 2 = 17l.6 (100-14-1) 5-Nitrosalicylic Acid 2-Hydroxy-5-nitrobenzoic acid;
Pale-yellow crystals, lachrymatory, practically insoluble in C 7H 5NO s = 183 (96-97-9)
water, very soluble in ethanol (96%). General reagent grade of commerce.
4-(4-Nitrobenzyl)pyridine Cl2HION202 = 214.2 (1083- N-Nitrosodiethanolamine 2,2 '-(Nitrosoimino )diethanol;
48-3) C 4H ION 2 0 3 = 134.1 (1116-54-7)
Yellow powder. Yellow liquid, miscible with anhydrous ethano!.
Melting point, about 70°. n~, about l.485; boiling point, about 125°.
Nitrochromic Reagent N-Nitrosodiisopropanolamine 1,1 '-
Dissolve 0.7 g of potassium dichromate in nitric acid and dilute (Nitrosoimino)bispropan-2-01;
to 100 mL with the same acid. C6Hl4N203 = 162.2 (53609-64-6)
Nitroethane C 2H s N0 2 = 75.1 (79-24-3) Boiling point, 122 0 to 124°.
Clear, oily, colourless liquid. Nitrosodipropylamine DipropyInitrosamine;
C 6 H l4 N 2 0 = 130.2 (621-64-7)
Boiling point, about 114 0 •
2-Nitroethanol C 2H sN0 3 = 91.1 (625-48-9) Liquid, soluble in anhydrous ethanol and in strong acids.
d~g, about 0.915; boiling point, about 78 •
0
General reagent of commerce.

Boiling point, about 205 0
•
Appropriate grade for chemiluminescence determination.
Nitrofurantoin CSH6N 40 S = 238.2 (67-20-9) Nitrosodipropylamine SoIution Inject 78.62 g of
anhydrous ethanol through the septum of a vial containing
Of the British Pharmacopoeia. nitrosodipropylamine. Dilute 1/100 in anhydrous ethanol and
(5-Nitro-2-furyl)methylene Diacetate Nitrofurfural place O.5-mL aliquots in crimp-sealed vials.
diacetate; 5-nitrofurfurylidene diacetate; Store in the dark at 5°.
C 9 H 9 N0 7 = 243 .2 (92-55-7)
0
Nitrotetrazolium Blue 3,3'-(3,3'-Dimethoxy-4,4'-
Yellow crystals; melting point, about 90 •
diphenylene) di [2- (4-nitrophenyl)-5-phenyl-2H-tetrazolium]
Nitrogen N 2 = 28.01 (7727-37-9) dichloride, p-nitro-tetrazolium blue;
Nitrogen, washed and dried. C4oH30CI2NIO06 = 818 (298-83-9)
Nitrogen Dioxide N0 2 = 46.01 (10102-44-0) Crystals, soluble in methanol, giving a c1ear, yellow solution.
0
Content, minimum 98.0% v/v. Melting point, about 189 with decomposition.
,
Nitrogen for Chromatography Nitrous Oxide N 2 0 = 44.01 (10024-97-2)

Content, minimum 99.95% v/v. Content, minimum 99.99 % v/v.
Nitrogen Gas Mixture Nitrogen containing 1% v/v of each Nitrogen monoxide, less than 1 ppm.
of the following gases: carbon dioxide R2, carbon monoxide Rl Carbon monoxide, less than 1 ppm.
and oxygen R1. Nitro-vanado-molybdic Reagent Nitro-molybdovanadic
Nitrogen Monoxide Nitric oxide; NO = 30.01 (10102-43- reagent
9)
Solution A Dissolve 10 g of ammonium molybdate in water,
Content, minimum 98.0% v/v. add 1 mL of ammonia and dilute to 100 mL with water.
Nitrogen, Oxygen-free Nitrogen which has been freed Solution B Dissolve 2.5 g of ammonium vanadate in hot
from oxygen by passage through alkaline pyrogallol solution. water, add 14 mL of nitn'c acid and dilute to 500 mL with
Nitrogen Rl water.
Content, minimum 99.999% v/v. To 96 mL of nitric acid add 100 mL of solution A and
Carbon monoxide, less than 5 ppm. 100 mL of solution B and dilute to 500 mL with water.
Oxygen, les s than 5 ppm. Nonivamide N-[(4-Hydroxy-3-
Nitromethane CH3 N0 2 = 6l.04 (75-52-5) methoxyphenyl)methyl]nonanamide;
C 17 H 27N0 3 = 293.4 (2444-46-4)
Clear, colourless, oily liquid, slightly soluble in water,
miscible with ethanol (96%). White or almost white, crystalline powder, practically
insoluble in cold water, freely soluble in anhydrous ethano!.
d~g, 1.132 to 1.134; n~, 1.381 to l.383.
Nonivamide used in the testfor nonivamide in the monograph
Distillation range (2.2. 11) Not less than 95% distils Capsicum (1859) complies with the following additional test.
0
between 100 and 103°.
Assay Liquid chromatography (2.2.29) as prescribed in the Oetanoic Acid Caprylic acid; C SH 160 2 = 144.2
monograph Capsieum (1859). (124-07-2)
Content, minimum 98.0%, calculated by the normalisation General reagent grade of commerce.
procedure. A colourless, oily liquidó boiling point, about 237°; weight
Nonylamine 1-Arninononane; per mL, about 0.92 g.
C 9H 21 N = 143.3 (120-20-9) Oetanol Caprylic alcohol, 1-0ctanol, Octan-l-ol;
Corrosive, colourless, clear liquido C SH 1S O = 130.2 (111-87-5)
dio, about 0.788; n~o, about 1.433. Colourless liquid, practically insoluble in water, miscible with
Noradrenaline Acid Tartrate Noradrenaline bitanrate; ethanol (96%).
CSHllN03, C4H606 = 319.3 (69815-49-2) dig, about 0.828; boiling point, about 195 0 •
General reagent grade of commerce. Oetan-l-ol See oetanol.
A white, crystalline powder; melting point, about 102°. Oetan-2-o1 see-Octyl alcohol; CSH1 SO = 130.2
Nordazepam 7-Chloro-2,3-dihydro-5-phenyl-1H-1,4- ( 6169-06-8)
benzodiazepin-2-one, desmethyldiazepam; General reagent grade of commerce.
C 1sH ll CIN 20 = 270.7 (1088-11-5) An oily liquidó boiling point, about 178°; weight per mL,
White or pale yellow, crystalline powder, practically insoluble about 0.82 g.
in water, slightly soluble in ethanol (96%). 3-0etanone Ethylpentylke1One; C SH 16 0 = 128.2 (106-
Melting point, about 216°. 68-3)
DL-Norleueine (RS)-2-Aminohexanoic acid; Colourless liquid with a characteristic odour.
C 6H 13 N0 2 = 131.2 (616-06-8) dig, about 0.822; ng>, about 1.415; boiling point, about
Shiny crystals, sparingly soluble in water and in ethanol 167°.
(96%), soluble in acids. 3-0ctanone used in gas ehromatography complies with the
Noroxymorphone C 16H 17N0 4 = 287.3 (33522-95-1) following additional test.
General reagent grade of commerce. Assay Gas chroma1Ography (2.2.28) as prescribed in the
Norpseudoephedrine Hydroehloride monograph Lavender oil (1338).
C 9H 13 NO,HCI = 187.7 (53643-20-2) Test soZution The substance to be examined.
General reagent grade of commerce. Content Minimum 98.0%, calculated by the nonnalisation
A crystalline powder; melting point, 180° 10 181 °. procedure.
Noseapine Hydroehloride Oetoxinol 10 r:J.- [4-( 1,1,3,3-Tetramethylbutyl)phenyl]-úJ-
C22H 23N07,HCI,H20 = 467.9 (912-60-7) hydroxypoly(oxyethylene); C34H62011 (average) = 647
(9002-93-1)
Of the British Phannacopoeia.
Clear, pale-yellow, viscous liquid, miscible with water, with
Oehratoxin A Solution 50Jlg/L solution of (2S)-2-
acetone and with ethanol (96%), soluble in 1Oluene.
([[ (3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-
2- benzopyran-7 -yl] carbonyl] amino )-3-phenylpropanoic acid S10re in an airtight container.
(Ochratoxin A) in a mixture of 1 volume of aeetie acid and Oetreotide Aeetate n-phenylalanyl-L-cysteinyl-L-
99 volumes of benzene. phenylalanyl-n-tryp1Ophyl-L-lysyl-L-threonyl-N-[(lR,2R)-2-
Oetadeean-l-ol Stearyl alcohol; C 1sH 3S O = 270.5 hydroxy-l-(hydroxymethyl)propyl]-L-cysteinamide cyclic
(112-92-5) (2 -> 7)-disulfide acetate; C 49 H 66 N 100 IOS2,XC2H402 = 1019
(acetate-free peptide) (83150-76-9), (79517-01-4)
Purified general reagent grade of commerce.
It contains a variable amount of acetic acid.
White f1akes or granules; melting point, about 58°.
Appearance, white or almost white powder.
Oetadeeyl [3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl]-propionate] C3sH 620 3 = 530.9 (2082- Solubility, freely soluble in water and acetic acid.
79-3) Content, minimum 96.0%.
White or slightly yellowish, crystalline powder, practically Oetylamine l-Amino-octane, octan-l-amine,
insoluble in water, very soluble in ace10ne and in hexane, n-octylamine; C SH 19N = 129.3 (111-86-4)
slightly soluble in methanol. Colourless liquido
Melting point, 49° to 55°. dig, about 0.782; boiling point, 175° 10 179°.
Oetana! Octyl aldehyde; C SH 160 = 128.2 (124-13-0) Olearnide (Z)-Octadec-9-enoamide; C 18 H 3S NO = 281.5
Oily, colourless liquid. Practically insoluble in water. Yellowish or white powder or granules, practically insoluble
Oetanal used in gas ehromatography eomplies with the following in water, very soluble in methylene chloride, soluble in
additional test. anhydrous ethanol.
Assay Gas chroma1Ography (2.2.28) as prescribed in the Melting point, about 80°.
monograph Sweet orange oil (1811) . Oleanolic Acid Astrantiagenin C, 3p-Hydroxyolean-12-en-
Content, minimum 99%, calculated by the nonnalisation 28-oic acid; C30H 4S0 3 = 456.7 (508-02-1)
procedure. Oleic Acid (9Z)-Octadec-9-enoic acid; ClsH340 2 = 282.5
Oetane n-Octane; C SH 18 = 114.2 (111-65-9) (112-80-1)
Analytical grade of commerce Clear, colourless liquid, practically insoluble in water.
n-Oetane See Oetane. d¡O, about 0.891; n~o, about 1.459; melting point, 13° to 14°.
Oleie acid used in the assay 01 total fatly acids in Saw palmetto Organosilica Polymer, Amorphous, Octadecylsilyl
fruit eomplies with the lollowing additional test. Synthetic, spherical hybrid particles, containing both
Assay Gas chromatography (2.2.28) as prescribed in the inorganic (silica) and organic (organosiloxanes) components,
monograph Saw palmetto fruit (1848) . chemically modified at the surface by trifunctionally bonded
Content, minimum 98%, ca1culated by the normalisation octadecylsilyl groups.
procedure. Organosilica Polymer, Arnorphous, OctadecyIsilyl,
Oleuropein 2-(3,4-DihydtOxyphenyl)ethyl [(2S,3E,4S)-3- End-capped
ethylidene-2-(b-D-glucopyranosyloxy)-5-(methoxycarbonyl)- Synthetic, spherical hybrid particles, containing both
3,4-dihydro-2H-pyran-4-yl] acetate; C25H32013 = 540.5 inorganic (silica) and organic (organosiloxanes) components,
(32619-42-4) chemically modified at the surface by trifunctionally bonded
Powder, soluble in methanol. octadecylsilyl groups. To minimise any interaction with basic
compounds, it is carefully end-capped to cover most of the
Oleuropein used in Olive Leal eomplies with the lollowing test.
remaining silanol groups. The particle size is indicated after
Assay Liquid chromatography (2.2.29) as prescribed in the the name of the reagent in the tests where it is used.
monograph Olive leal (1878) .
Organosilica Polymer, Amorphous, Polar-embedded
Content, minimum 80%, ca1culated by the normalisation Octadecylsilyl, End-capped
procedure.
Synthetic, spherical hybrid particles containing both inorganic
Oleyl Alcohol (9Z)-octadec-9-en-I-ol; C 1sH 36 0 = 268.5 (silica) and organic (organosiloxanes) components,
(143-28-2) chemically modified at the surface by the bonding of polar-
Boiling point, about 207°; n~, 1.460. embedded octadecylsilyl groups. To minimise any interaction
Content, minimum 85%. with basic compounds, it is carefully end-capped to cover
Olive Oil (8001-25-0) Virgin Olive Oil of the British most of the remaining silanol groups. The particle size is
Pharmacopoeia. indicated after the name of the reagent in the tests where it is
Olive Oil Substrate Emulsion Homogenise 40 mL of used.
olive oil, 330 mL of acacia solution and 30 mL of water in an Organosilica Polymer, Arnorphous, Propyl-2-
800-mL beaker placed in a vessel containing a mixture of ice phenylsilyl, End-capped
and ethanol as cooling mixture. Emulsify using a mixer at an Synthetic, spherical hybrid particles containing both inorganic
average speed of 1000 to 2000 revolutions per minute. Cool (silica) and organic (organosiloxanes) components,
to 5° to 10°. Increase the mixing speed to 8000 revolutions chemically modified at the surface by the bonding of propyl-
per minute. Mix for 30 minutes keeping the temperature 2-phenylsilyl groups. To minimise any interaction with basic
below 25° by the continuous addition of crushed ice into the compounds, it is carefully end-capped to cover most of the
cooling mixture (a mixture of ca1cium chloride and crushed remaining silanol groups. The particle size is indicated after
ice is also suitable) . Store this preparation (the stock the name of the reagent in the tests where it is used.
emulsion) in a refrigerator and use within 14 days. The Organosilica Polymer for Mass Spectrometry,
emulsion must not separate into two distinct layers. Check Arnorphous, Octadecylsilyl, End-capped
the diameter of the globules of the emulsion under a Synthetic, spherical hybrid particles containing both inorganic
microscope. At least 90% have a diameter below 3 J-lm and (silica) and organic (organosiloxanes) components. To
none has a diameter greater than 10 J-lm. Shake the emulsion minimise any interaction with basic compounds, it is carefully
thoroughly before preparing the substrate emulsion. end-capped to cover most of the remaining silanol groups.
For 10 determinations mix the following solutions in the The particle size is indicated after the name of the reagent in
order indicated: 100 roL of the stock emulsion, 80 mL of the tests where it is used.
tris-ehloride buffer solution, 20 mL of a freshly prepared Orthophosphoric Acid Phosphoric acid;
8% w/v solution of sodium tauroeholate EPBRP and 95 mL of H 3P04 = 98.0 (7664-38-2)
water.
Use on the day of preparation.
than 84% w/w ofH 3P0 4 and about 15.7M in strength.
Oracet Blue B Solvent blue 19
A corrosive liquid; weight per mI, about 1.75 g.
A mixture of l-methylamino-4-anilinoanthraquinone
Orthophosphorous Acid See phosphorous acid.
(C21HI6N202) and l-amino-4-anilinoanthraquinone
(C2oHI 4N20 2). When used for titration in non-aqueous Osmium Tetroxide Osmic acid; OS04 = 254.2
media, it changes from blue (basic) through purple (neutral) (20816-12-0)
to pink (acidic). Light-yellow needles or a yellow, crystalline mass,
Oracet Blue B Solution A 0.5% w/v solution of oraeet hygroscopic, light sensitive, soluble in water and in ethanol
blue B in anhydrous aeetie acid. (96%) .
Oracet Blue 2R l-Amino-4-(phenylamino)anthracene- Store in an airtight container.
9,10-dione; CI 61110; C2oH¡ 4N202 = 314.3 (4395-65-7) Osmium Tetroxide Solution A 0.25 % w/v solution in
Melting point, about 194°. 0.05M sulfurie acid.
Osthole 7-Methoxy-8-(3-methylbut-2-enyl)-2H-l-
Oracet Blue 2R Solution A 0.5% w/v solution of oraeet blue
benzopyran-2-one; 7-Methoxy-8-isopenteny1coumarin;
2R in anhydrous aeetie acid.
C¡SH¡ 60 3 = 244.3 (484-12-8)
Orcinol 5-Methylbenzene-l,3-diol monohydrate;
C 7 H s0 2,H 20 = 142.2 (6153-39-5)
Oxalic Acid Ethanedioic acid dihydrate;
Crystalline powder, sensitive to light. C 2H 20 4 ,2H 20 = 126.1 (6153-56-6)
Boiling point, about 290°; melting point, 58° to 61 0 . White or almost white crystals, soluble in water, freely
soluble in ethanol (96%) .
Oxalic Acid and Sulfuric Acid Solution Oxalic acid and Palmitoleic Acid (9Z)-Hexadec-9-enoic acid;
sulphuric acid solution. C16H3002 = 254.4 (373-49-9)
A 5% w/v solution of oxalic aeíd in a cooled mixture of equal Clear, colourless liquido Boiling point, about 162°.
volumes of sulfurie acid and water. Palmitoleie acid used in the assay of total fatty acids in Saw
Oxazepam (604-75-1) palmetto fruit complíes with the following test.
Of the British Pharmacopoeia. Assay Examine by gas chromatography (2 .2.28) as
Ox Brain, Acetone-dried Cut into small pieces a fresh prescribed in the monograph on Saw palmetto fruit.
ox brain previously freed from vascular and connective The content of palmitoleic acid is not less than 98%,
tissue. Place in acetone for preliminary dehydration. calculated by the normalisation procedure.
Complete the dehydration by pounding in a mortar 30 g of Palmityl Alcohol Cetyl alcohol. 1-Hexadecanol;
this material with successive quantities, each of 75 mL, of C 16H 340 = 242.4 (36653-82-4)
aeetone until a dry powder is obtained after filtration. Dry at Melting point, about 48'.
37° for 2 hours or until the odour of acetone is no longer
presento General reagent grade of commerce containing a minimum
of 96% of C 16H 34 0.
2,2' -Oxybis (N, N-dimethylethylamine)
Pancreas Powder Of the British Pharmacopoeia.
bis(2-Dimethylaminoethyl) ether; CsHzoNzO = 160.3
(3033-62-3) Papain (9001-73-4)
Colourless, corrosive Iiquid. A proteolytic enzyme obtained from the latex of the green
fruit and leaves of Cariea papaya L.
d~g, about 0.85; nfi!, about 1.430.
Oxygen Oz = 32.00 (7782-44-7) Papaverine Hydrochloride Of the British
Pharmacopoeia.
Content, minimum 99.99% v/v.
Paper Chromatography Performance Test Solutions
Nitrogen and argon, les s than 100 ppm.
Test solution (a) Sodium pertechnetate (l 9m Te) injection
Carbon dioxide, less than 10 ppm. (fission) (0124) or Sodium pertechnetate (l 9m Te) injeetion (non-
Carbon monoxide, les s than 5 ppm. fission) (0283).
Oxygen Rl O2 = 32.00 Test solution (b) In a c10sed vial mix 100 f1L of a 5 gIL
Content, minimum 99% v/v. solution of stannous chlonde in 0.05 M hydroehlorie aeid and
Oxytetracycline Hydrochloride Of the British 100 MBq to 200 MBq of Sodium pertechnetate (l 9m Te)
Pharmacopoeia. injection (fission) (0124) or Sodium pertechnetate (l'hnTe)
injection (non-fission) (0283) in a volume not exceeding
Palladium Pd = 106.4 (7440-05-3)
2 mL.
Paper for Chromatography
Palladium Chloride See Palladium(Il) ehloride.
Pure cellulose grade thin paper with a smooth surface and a
Palladium(I1) Chloride Palladous chloride; thickness of about 0.2 mm.
PdClz = 177.3 (7647-10-1)
Chromatographic separation T o 2 strips of paper for
General reagent grade of commerce containing not less than chromatography apply separately 2-5 flL of test solution (a)
59% ofPd. and test solution (b) of paper chromatography performance test
A hygroscopic, brownish red powder or red crystals; melting solutions. Develop over a pathlength of 3/4 of the paper
point, 678 to 680°.
0
height, using a mixture of equal volumes of methanol and
Palladium Chloride Solution Dissolve 1 g of water. Allow to dry and determine the distribution of
palladium(Il) ehloride in 10 mL of warm hydrochloric acid. radioactivity using a suitable detector. The paper is not
Dilute the solution to 250 mL with a mixture of equal satisfactory, unless the chromatogram obtained with test
volumes of 2M hydrochloric acid and water. Dilute this solution solution (a) shows a single radioactivity spot with an R¡ value
immediately before use with 2 volumes of water. in the range 0.8-1.0 and the chromatogram obtained with
Palmatine Chloride Berbericine Chloride; C 21 H 22 N0 4,CI test solution (b) shows a single radioactivity spot at the
= 387.86 (10605-02-4) application point (R¡value in the range 0.0-0.1).
General reagent grade of commerce. Paracetamol Of the British Pharmacopoeia.
Palmitic Acid Hexadecanoic acid; Paracetamol, 4-Aminophenol-free Paracetamol of the
C16H320Z = 256.4 (57-10-3) British Pharmacopoeia or paracetamol that has been
recrystallised from water and dried at a pressure of 2 kPa at
70°. Repeat the procedure until it complies with the
White, crystalline scales; melting point, about 63°. following test.
Complíes with the following test. Dissolve 5 g of the dried material in sufficient methanol
Homogeneity Carry out test A for Identification described (50%) to produce 100 mL, add I mL of a freshly prepared
in the monograph for Chloramphenicol Palmitate applying to solution containing 1% w/v of each of sodium nitroprusside and
the plate 4 flL of a 0.2% w/v solution in acetone. The anhydrous sodium carbonate, mix and allow to stand for
chromatogram shows only one principal spot. 30 minutes protected from light. No blue or green colour is
Palmitic acid used in [he assay of total fatty acids in Saw produced.
palmetto fruit complíes with the following additional test. Paraffin, Liquid Liquid Paraffin of the British
Assay Examine by gas chromatography (2.2.28) as Pharmacopoeia.
prescribed in the monograph on Saw palmetto fruit. Paraffin, White Soft A semi-liquid mixture of
The content of palmitic acid is not less than 98%, calculated hydrocarbons obtained from petroleum and bleached.
by the normalísation procedure. Paraldehyde (123-63-7) Of the British Pharmacopoeia.
Pararosaniline Hydrochloride CI 42500; pararosaniline n-Pentane Pentane; CSH 10 = 72.15 (109-66-0)

chloride; basic red 9; C1 9H1 SCIN3 = 323.8 (569-61-9) General reagent grade of commerce.
General reagent grade of commerce. A colourIess, volatile liquid; boiling point, about 36°; d~g,
A bluish red, crystalline powder; melting point, about 270°, about 0.63; n~, about 1.359.
with decomposition. Pentane used in spectrophotomerry complies with the following test.
Pararosaniline Solution, Decolorised To 0.1 g of Transmittance Not les s than 20% at 200 nm, 50% at
pararosaniline hydrochloride add 60 mL of water and a solution 210 nm, 85% at 220 nm, 93 % at 230 nm and 98% at
of 1.0 g of anhydrous sodium sulfite or 2.0 g of sodium sulfite 240 nm, Appendix II B, determined using water in the
or 0.75 g of sodium metabisulfite in 10 mL of water. Add reference cel!.
slowly, with stirring, 6 mL of 2M hydrochloric acid, stopper 1,2-PentanedioI (2RS)-Pentane-l,2-diol;
the ftask and continue stirring until completely dissolved. C SH 12 0 2 = 104.2 (5343-92-0)
Dilute to 100 mL with water. Allow to stand for 12 hours
before use. dio, about 0.971 ; n~, about 1.439; boiling point, about
201 °.
Pentanol See Pentan-1-ol.
Parthenolide (E)-(5S,6S)-4,5-Epoxygermacra-
1 (10),11 (13)-dieno-12(6)-lactone; 3-Pentanone Diethyl Ketone; CSHlOO = 86 .13 (96-22-0)
ClsHzo0 3 = 248.3 (20554-84-1 ) Pentan-l-ol n-Penryl alcohol; Pentanol;
C SH 120 = 88.15 (71-41-0)
General reagent grade of cornmerce.
[a]j32, - 71.4 (in a 0.22% w/v solution in dichloromethane);
melting point, 115° to 116°. A colourless liquid; boiling point, about 137°; n~, about
Assay Carry out the Assay described in the monograph on 1.410.
Feverfew, at the concentration of the reference solution. The Pentetic Acid
content of parthenolide is not les s than 90% by normalisation. [[ (Carboxyrnethyl)imino] bis (ethylenenitrilo)1tetraacetic acid;
Penicillinase Solution Dissolve 10 g of casein C14H23N 301O = 393.3 (67-43-6)
hydrolysate, 2.72 g of potassium dihydrogen orthophosphate and Appearance, white or almost white powder.
5.88 g of sodium citrate in 200 mL of water, adjust the pH to Solubiliry, slightly soluble in water.
7.2 with 5M sodium hydroxide and dilute to 1000 mL with Melting point, 219° to 220°, with decomposition.
water. Dissolve 0.41 g of magnesium sulfate in 5 mL of water tert-Pentyl Alcohol 2-Methylbutan-2-ol; tert-amyl alcohol;
and add 1 mL of a 0.16% w/v solution of ammonium iron(Il)
CSH1 ZO = 88.1 (75-85-4)
sulfate and sufficient water to produce 10 rnL. Sterilise both
solutions by heating in an autoclave, cool, mix, distribute in General reagent grade of commerce.
shallow layers in conical flasks and inoculate with Bacillus Not less than 95% distils between 100 and 104°; d~g, about
0
cereus (NCTC 9946). Incubate the flasks at 18° to 37° until 0.81.
growth is observed and then maintain at 35° to 3r for 16 Store protected from light.
hours, agitating constantly to ensure maximum aeration. Pepsin A substance containing a proteolytic enzyme of the
Centrifuge and sterilise the supematant fluid by filtration gastric secretion of animals, diluted, if necessary, by
through a suitable membrane filter. admixture with Lactose or Sucrose. Use a grade of
1.0 mL of Penicillinase Solution should hydrolyse commerce capable of digesting 2500 times its own weight of
benzylpenicillin to benzylpenicilloic acid at the rate of at least coagulated egg albumen.
500 mg per hour at 30° and pH 7, provided that the Pepsin Powder (9001-75-6) Of the British
concentration of benzylpenicillin does not fall below the level Pharmacopoeia.
necessary for enzyme saturation. The Michaelis constant for
Perchloric Acid HCI0 4 = 100.5 (7601-90-3)
benzylpenicillin of the penicillinase in penicillinase solution is
approximately 12 J..lg per mL. When no molarity is indicated, use analytical reagent grade
of cornmerce containing not less than 70.0% and not more
Sterility Complies with the test for sterility, than 73 .0% w/w ofHCl0 4 and about 12M in strength; a
Appendix XVI A.
corrosive liquid; weight per mL, about 1.7 g.
Store at 0° to 2° and use within 2 or 3 days. When freeze
For 9M perchloric acid use analytical reagent grade solution
dried and kept in sealed ampoules it may be sto red for
of cornmerce containing about 60% w/w of HCI0 4 ; weight
several months.
per mL, about 1.54 g.
PentaerythrityI Tetrakis[3-(3,5-di-tert-butyI-4-
Solutions of any other molarity XM should be prepared by
hydroxyphenyI)propionate] C73HlOS01Z = 1178 (6683-
diluting 82x mL of perchloric acid with water to 1000 mL.
19-8)
Perchloric Acid SoIution Dilute 8.5 mL of perchloric acid
General reagent grade of commerce. to 100 mL with water (about 1M).
A white to slightly yellowish, crystalline powder; melting Periodic Acetic Acid SoIution Dissolve 0.446 g of
point, 110° to 125°; cx-form, 120° to 125°; ~-form, 110° to sodium periodate in 2.5 mL of sulfuric acid (25%) and dilute
115°. to 100 mL with glacial acetic acid.
Pentafluoropropanoic Acid Periodic Acid H sI0 6 = 227 .9 (10450-60-9)
C 3 HF sOz = 164.0 (422-64-0) General reagent grade of commerce.
Clear, colourIess liquid; d~g, about l.561; n~, about 1.284; Melting point, about 122°.
boiling point, about 97°. Periodic Acid Reagent Dissolve 0.5 g of sodium periodate
Pentafluoropropionic Anhydride in 5 mL of water, add 1 mL of 2M sulfuric acid and dilute to
C 6 F lO 0 3 = 310.0 (356-42-3) 10 mL with water.
Pentane See n-Pentane. Prepare irnmediately before use.
Periodic Acid Solution General reagent grade of Petroleum Spirit (boiling range, 40° to 60°), Aromatic-
commerce containing about 50% w/v of HI0 4,2H 20 . free Petroleum spirit (boiling range, 400 to 600) complying
Permethrin C21H20Cl203 = 391.3 (52645-53-1) with the following additional test.
Use a grade of commerce suitable for pesticide residue Light absorption Absorbance against air at 235 nm, not
analysis. A suitable certified reference solution (10 ng/JlL in more than 0.7, Appendix JI B.
cyclohexane) roay be used. pH Indicator Strip Plastic strip containing multiple
Melting point, 34° to 35°. segments of different dye-impregnated papers allowing visual
Peroxide Test Strips determination of pH in the prescribed range by comparison
with a master chart.
Use commercial test strips with a suitable scale in the range
CJ.- Phellandrene (R)-5- Isopropyl-2-methyl-cyc1ohexa-l ,3-
from O ppm to 25 ppm peroxide.
diene;C 10 H 16 = 136.2 (4221-98-1)
Peroxyacetic Acid Solution Dilute 1 mL of hydrogen
peroxide (100 volumes) to 100 mL with glacial acetic acid. Mix General reagent grade of commerce.
and allow to stand for 12 hours before use. d~g, about 0.839; n~, about 1.471; [albO, about - 217;
Discard 24 hours after preparation. boiling point, 171 ° to 174°.
Perylene Dibenz [de,k~anthracene; CI.-Phellandrene used in gas chromatography complies with the
following test.
C 2o H 12 = 252.3 (198-55-0)
General reagent grade of commerce. Assay Liquid chromatography as described in the
monograph for Eucalyptus Oil using the reagent being
examined as the test solution. Minimum of 95.0% calculated
Petroleum, Light Petroleum ether 50-70°; (8032-32-4) by normalisation.
A clear, colourless, fiammable liquid without fiuorescence, Phenacetin p-Ethoxyacetanilide;
practically insoluble in water, miscible with ethanol (96%). C lOH 13 N0 2 = 179.2 (62-44-2)
dig, 0.661 to 0.664. General reagent grade of commerce.
Distillation range (2.2. 11) 50° to 70°. Melting point, about 135°.
Petroleum Rl, Light Petroleum ether 40-60 oC. Phenanthrene C 14H lO = 178.1 (85-01-8)
Complies with the requirements prescribed for light petroleum, General reagent grade of commerce.
with the following modifications. Melting point, about 100°.
d~g, 0.630 to 0.656. Phenanthroline Hydrochloride
Distillation range (2.2.11) 40° to 60°. C12HsN2,HCI,H20 = 234.7 (3829-86-5)
It does not become cloudy at O°c. General reagent grade of commerce.
Petroleum Rl, Light Petroleum ether 30-40°. A white or almost white powder; melting point, about 215°,
Complies with the requirements prescribed for light petroleum, with decomposition.
with the following modifications. Phenazone Of the British Pharmacopoeia.
d~g, 0.620 to 0.630. Phenol Of the British Pharmacopoeia.
Distillation range (2.2. 11) 30° to 40°. Phenol, Liquefied
It do es not become cloudy at O°. General reagent grade of commerce.
Petroleum R3, Light Petroleum ether 100-120°. A solution of phenol in water containing about 80% w/w of
Complies with the requirements prescribed for light petroleum, C6H 6 0.
with the following modifications. Phenol Red 4,4' -(3H-2,l-Benzoxathiol-3-ylidene)diphenol
d~g, about 0.720. S,S-dioxide; phenolsulfonphthalein;
Distillation range (2.2. 11) 100° to 120°. C19H140 SS = 354.4 (143-74-8)
Water (2.5. 12) maximum 0.03%. General reagent grade of cornrnerce.
Petroleum R4, Light Petroleum ether 80° to 100°. Produces a yellow colour in neutral and very faintly acidic
Complies with the requirements prescribed for light petroleum, solutions and a red colour in weakly alkaline solutions.
with the following modifications. Phenol Red Solution Dissolve 0.1 g of phenol red in
2.82 mL of O.IM sodium hydroxide and 20 mL of ethanol
Distillation range (2.2.11),80° to 100°; d~g, about 0.70.
(96%) and add sufficient water to produce 100 mL.
Petroleum Spirit Petroleum ether; light petroleum
Analytical reagent grades of commerce.
Sensitivity A mixture of 0.1 mL and 100 mL of carbon
Colourless, volatile, highly fiammable liquids obtained from dioxide-free water is yellow. Not more than 0.1 mL of
petroleum, consisting of a mixture of the lower members of 0.02M sodium hydroxide VS is required to change the colour
the paraffin series of hydrocarbons supplied in the following of the solution to reddish vio let.
fractions:
Colour change pH 6.8 (yellow) to pH 8.4 (reddish
boiling range, 30° to 40°; weight per mL, about 0.63 g, violet).
boiling range, 40° to 60°; weight per mL, about 0.64 g, Phenol Red Solution Rl Buffered phenol red solution
boiling range, 50° ro 70°; weight per mL, about 0.66 g, Dissolve 33 mg of phenol red in 1.5 mL of 2M sodium
boiling range, 60° to 80°; weight per mL, about 0.67 g, hydroxide and dilute to 100 mL with water (solution A) .
boiling range, 80° to 100°; weight per mL, about 0.70 g, To 250 mL of 2M sodium hydroxide add 325 mL of 2M acetic
boiling range, 100° to 120°; weight per mL, about 0.72 g, acid and 575 roL of water (solution B). Mix 25 mL of
solution A with 475 mL of solution B.
boiling range, 120° to 160°; weight per mL, about 0.75 g.
V-AlOO Appendix 1 A 2014
Phenol Red Solution R2 described in the monograph for Phenoxymethylpenicillin

Solution 1 Dissolve 33 mg of phenol red in 1.5 mL of applying ro the plate 10 ¡.¡L of a 0.010% w/v solution in
2M sodium hydroxide and dilute to 100 mL with water. methanol (50%). The chromarogram shows only one
principal spot.
Solution II Dissolve 25 mg of ammonium sulfate in 235 mL
of water, add 105 mL of 2M sodium hydmxide and 135 mL of 2-Phenoxyaniline 2-Phenoxybenzenamine; 2-aminophenyl
2M acetic acid. phenyl ether; C l2H Il NO = 185.2 (2688-84-8)
Add 25 mL of solution 1 to solution !l. If necessary, adjust Phenoxybenzamine Hydrochloride N-(2-Chloroethyl)-
the pH of the mixture ro 4.7. N-(l-methyl-2-phenoxyethyl) benzylamine hydrochloride;
C 1sH 23 ClzNO = 340.3 (63-92-3)
Phenol Red Solution R3
Solution 1 Dissolve 33 mg of phenol red in 1.5 mL of
2M sodium hydroxide and dilute to 50 mL with water. Melting point, about 138°.
Solution II Dissolve 50 mg of ammonium sulfate in 235 mL Phenoxyethanol See 2-Phenoxyethanol.
of water, add 105 mL of 2M sodium hydmxide and 135 mL of 2-Phenoxyethanol Phenoxyethanol;
2M acetic acid. C SH IO 0 2 = 138.2 (122-99-6)
Add 25 mL of solution 1 ro solution !l and adjust the pH of General reagent grade of commerce.
the mixture to 4.7, ifnecessary. di8,
A colourless, oily liquid; about 1.11 ; n~, about 1.537;
Phenoldisulfonic Acid Solution Phenoldisulphonic acid freezing point, not les s than 12 0 •
solution Phenyl Benzoate C 13H IO 0 2 = 198.2 (93-99-2)
A clear liquid which may develop a pale brown colour on General reagent grade of commerce.
storage, prepared either by heating 3 g of phenol with 20 mL Melting point, about 70°.
of sulfuric acid on a water bath for 6 hours and transferring
PhenylIsothiocyanate C 7 H sNS = 135.2 (103-72-0)
the resulting liquid ro a stoppered vessel, or by diluting a
25% w/v solution of commerce with sulfuric acid to contain Use a grade of commerce suitable for protein sequencing.
15% w/v ofphenol. The solution complies with the following di8, about 1.13; n~o, about 1.65; boiling point, about 221 °.
test. Phenylacetic Acid CsH sO z = 136.2 (103-82-2)
Sensitivity to nitra te Evaporate a solution containing General reagent grade of commerce.
0.1 mg of potassium nitrate ro dryness in a porcelain dish on Boiling point, about 265°; melting point, about 75°.
a water bath. To the cooled residue add 1 mL of the reagent
and allow to stand for 10 minutes. Add 10 mL of water, Phenylalanine Of the British Pharmacopoeia.
cool, add 10 mL of 5M ammonia and dilute ro 25 mL with L-Phenylalanine See Phenylalanine
water. A distinct yellow colour is produced when compared o-Phenylbenzoic Acid 2-Biphenylcarboxylic acid;
with a solution prepared in the same manner but omining C 13H IO 0 2 = 198 (947-84-2)
the potassium nitrate. General reagent grade of commerce.
Phenolphthalein 3,3-Bis(4-hydroxyphenyl)phthalide; Melting point, about 113°.
C2oHl404 = 318.3 (77-09-8)
(E)-4-Phenylbut-3-en-2-one Benzalacerone;
General reagent grade of commerce. CIOHIOO = 146.2 (1896-62-4)
Phenolphthalein Paper Immerse strips of filter paper for General reagent grade of commerce.
a few minutes in phenolphthalein solution and allow to dry.
Phenolphthalein Solution
N-Pheny1carbazole C 1sH 13 N = 243 .3 (1150-62-5)
Dissolve 0.1 g of phenolphthalein in 80 mL of alcohol and
A white to pale tan, crystalline powder; melting point, about
Complies with the following tests.
96°.
Test/or sensitivity To 0.1 mL of the phenolphthalein
p- Phenylenediarnine Dihydrochloride 1,4-
solution add 100 mL of carbon dioxide-jree water. The
Diaminobenzene dihydrochloride;
solution is colourless. Not more than 0.2 mL of 0.02 M
C 6 H sN z,2HCI = 181.1 (615-28-1)
sodium hydroxide is required to change the colour to pink.
Colour change pH 8.2 (colourless) to pH 10.0 (red)
A white to pale tan, crystalline powder, tuming pink on
Phenolphthalein Solution Rl A 1.0% w/v solution of
exposure to airo
phenolphthalein in ethanol (96%) .
a -Phenylglycine See DL-Phenylglycine.
Phenolphthalein-Thymol Blue Solution Dissolve 0.1 g
of thymol blue in a mixture of 2.2 mL of O.IM sodium D-Phenylglycine (2R)-2-Amino-2-phenylacetic acid;
hydroxide and 50 mL of ethanol (96%) and dilute ro 100 mL CSH9NOZ = 151.2 (875-74-1)
with water. Mix 3 volumes of this solution with 2 volumes of Contains not less than 99 % of C SH 9N0 2.
phenolphthalein solution. White or almost white, crystalline powder.
Phenoxyacetic Acid 2-Phenoxyethanoic acid; DL-Phenylglycine 2-Amino-2-phenylacetic acid;
C SH S 0 3 = 152.1 (122-59-8) C SH 9N0 2 = 151.2 (2835-06-5)
White or almost white crystals; melting point, about 98°. A white, crystalline solid; melting point, about 290°, with
Complies with the following test. sublimation.
Homogeneity Examine under the conditions and at the Phenylhydrazine C 6 H sN 2 = 108.1 (100-63-0)
concentration prescribed in the test for Phenoxyacetic acid General reagent grade of commerce.
A colourless or yellowish liquid turning yellow or dark red on supernatant liquid add 5 volumes of acetone, centrifuge,
exposure to light and air; freezing point, not less than 18°. discard the supernatant liquid, dry the precipitate and store
Store protected from light and distil under reduced pressure protected from light in a vacuum desiccator.
before use. Phosphomolybdic Acid Dodecamolybdophosphoric acid;
Phenylhydrazine Hydrochloride Phenylhydrazinium approximately H 3 P0 4 ,12Mo0 3 ,24H 20 = 2258 (51429-74-4)
chloride; C 6 H sN 2,HCI = 144.6 (59-88-1) Analytical reagent grade of commerce.
Analytical reagent grade of commerce. Fine, orange-yellow crystals.
A white or almost white, crystalline powder, becoming brown Phosphomolybdic Acid Solution To 40 mL of a
on exposure to airó melting point, about 245°, with 10% w/v solution of phosphomolybdic acid add, cautiously
decomposition. and with cooling, 60 mL of sulfuric acid.
Store protected from light. Prepare immediately before use.
Pheny1hydrazine Hydroch1oride Solution Strong Phosphomolybdic Acid Solution, Ethanolic A 20% w/v
phenylhydrazine hydrochloride solution solution of phosphomolybdic acid in ethanol (96%) .
Dissolve 0.9 g of phenylhydrazine hydrochloride in 50 mL of Prepare irnmediately before use.
water. Decolorise with activated charcoal and filter. To the Phosphomo1ybdotungstic Reagent Lithium and sodium
filtrate add 30 mL of hydrochloric acid and dilute to 250 mL molybdotungstophosphate solution.
with water. Folin Ciocalteau phenol reagent of commerce.
Pheny1hydrazine-Sulfuric Acid Solution Dissolve 100 g of sodium tungstate and 25 g of sodium
Phenylhydrazine-sulphuric acid solution
molybdate in 700 mL of water, add 100 mL of hydrochloric
Dissolve 65 mg of phenylhydrazine hydrochloride, previously acid and 50 mL of orthophosphoric acid and heat the mixture
recrystallised from ethanol (85%), in sufficient of a mixture of under a refiux condenser for 10 hours. Add 150 g of lithium
170 volumes of sulfuric acid and 80 volumes of water to sulfate, 50 mL of water and 0.2 mL of bromine and boil to
produce 100 mL. remove excess bromine (about 15 minutes), cool, dilute to
Prepare irnmediately before use. 1000 mL with water and filter. The reagent should be yellow
l-Pheny1piperazine C JO H 14N 2 = 162.2 (92-54-6) in colour. If it acquires a greenish tint, it is unsatisfactory for
use but may be regenerated by boiling with 0.2 mL of
bromine. Care must be taken to remove excess bromine by
d~o, about 1.07; n~, about 1.588. boiling.
Phloroglucide 2,3',4,5' ,6-Biphenylpentol; Store at 2° to 8°.
C 12H IO 0 5 = 234.2 (491-45-2)
Phosphomolybdotungstic Reagent, Dilute Dilute
White or almost white powder, hygroscopic, light sensitive. 1 volume of phosphomolybdotungstic reagent with 2 volumes of
Slowly discolours on exposure to light. water.
Phloroglucino1 Benzene-1,3,5-triol; Phosphoric Acid Of the British Pharmacopoeia.
C 6 H 6 0 3 ,2H 20 = 162.1 (6099-90-7)
Phosphoric Acid, Dilute Dilute Phosphoric Acid of the
Analytical reagent grade of cornmerce. British Pharmacopoeia.
White or pale cream crystals; melting point, about 223°. Phosphoric Acid, Dilute Rl Dilute 93 mL of dilute
Phloroglucinol Solution To 1 mL of a 10% w/v solution phosphoric acid to 1000 mL with water.
of phloroglucinol in ethanol (96%) add 9 mL of hydrochloric Phosphorous Acid H 3 P0 3 = 82.0 (13598-36-2)
acid.
White or almost white, very hygroscopic and deliquescent
Store protected from light. crystalline mass; slowly oxidised by oxygen (air) to H 3 P0 4 .
Phosalone C12H15CIN04PS2 = 367.8 (2310-17-0) Unstable, orthorhombic crystals, soluble in water, in ethanol
Use a grade of commerce suitable for pesticide residue (96%) and in a mixture of 3 volumes of ether and 1 volume
analysis. A suitable certified reference solution (10 ng/¡.tL in of ethanol (96%).
iso-octane) may be used. d~l : 1.651; melting point, about 73°.
Melting point, 45° to 48°. Phosphorus Pentoxide Diphosphorus pentoxide;
Phospholipid Wash a quantity ofhuman or bovine brain P 2 0 S = 142.0 (1314-56-3)
freed from meninges and blood vessels and macerate in a Use a grade specially supplied for use in desiccators.
suitable blender. Weigh 1000 to 1300 g of the macerate and
A white, amorphous, deliquescent powder hydrated by water
measure its volume (V mL). Extract with three quantities,
with the evolution of heat.
each of 4 V mL, of acetone, filter by suction and dry the
precipitate at 3T for 18 hours. Extract the dried precipitate Store in an airtight container.
with rwo quantities, each of 2 V mL, of a mixture of Phosphotungstic Acid Solution Dissolve 10 g of sodium
2 volumes of petroleum spirit (boiling range, 30° to 40°) and tungstate in 75 mL of water and add 8 mL of orthophosphoric
3 volumes of petroleum spirit (boiling range, 40° to 600), acid. Heat under a refiux condenser for 3 hours, allow to
filtering each extract through a filter paper previously washed cool, filter and add sufficient water to produce 100 mL.
with the petroleum spirit mixture. Combine the extracts and Phtha1a1dehyde C SH 6 0 2 = 134.1 (643-79-8)
evaporate to dryness at 45° at a pressure not exceeding 0.7 General reagent grade of commerce.
kPa. Dissolve the residue in 0.2 V mL of ether and allow to
stand at 4° until a deposit is produced. Centrifuge and
evaporate the c1ear supernatant liquid under reduced Store protected from light and airo
pressure until the volume is about 100 mL per kg of the Phthalaldehyde Reagent Dissolve 2.47 g of boric acid in
original macerate. Allow to stand at 4° until a precipitate is 75 mL of water, adjust the pH to 10.4 with a 45% w/v
produced (12 to 24 hours) and centrifuge. To the c1ear solution of potassium hydroxide and add sufficient water to
produce 100 mL. Dissolve 1.0 g of phthalaldehyde in 5 mL of cx-Pinene (lR,5R)-2,6,6-Trimethylbicyclo[3.1.1]heptane;

methanol, add 95 mL of the boric acid solution and 2 mL of ClOH 1ó = 136.2 (7785-70-8)
mercaptoacetic acid and adjust the pH to 10.4 with the General reagent grade of commerce.
potassium hydroxide solution. dig, about 0.859; nf,°, about 1.466; boiling point,
Store protected from light and use within 3 days of 154° to 156°.
preparation. cx-Pinene used in gas chromatography complíes with the following
Phtha1azine C s H óN 2 = 130.1 (253-52-1) test.
General reagent grade of commerce. Assay Examine by gas chromatography as described in the
Melting poim, 89 c to 92°. monograph for Bitter-orange-flower Oil using the reagent
Phthalein Purple Metalphthalein; C32H 32N201 2 + aq being examined as the test solution. The area of the
(2411-89-4) principal peak is not less than 99.0% of the total area of the
peaks.
Indicator grade of commerce.
~- Pinene 6,6-Dimethyl-2-methylenebicyclo [3 .1.1]heptane;
A creamy white to brown powder complying with the
ClOH 1ó = 136.2 (127-91-3)
following test.
A colourless, oily liquid, odour reminiscent of turpentine,
Sensitivity Dissolve 10 mg in 1 mL of 13.5M ammonia
practically insoluble in water, miscible with ethanol (96%).
and dilute to 100 mL with water. To 5 mL of the solution
add 95 mL of water, 4 mL of 13.5M ammonia, 50 mL of fJ-Pinene used in gas chromatography complies with the following
ethanol (96%) and 0.1 mL of O.lM barium chloride VS; test.
the solution is bluish violet. Add 0.15 mL ofO.1M disodium Assay Examine by gas chromatography (2.2.28) as
edetate VS; the solution becomes colourless. prescribed in the monograph Bitter-orange-fiower oil (1175),
Phthalic Acid Benzene-1,2-dicarboxylic acid; using the substance to be examined as the test solution.
C SH ó0 4 = 166.1 (88-99-3) Content, minimum 99.0%.
General reagent grade of commerce. Piperazine Dipicrate Solution Dissolve 0.2 g of
A white, crystalline powder. piperazine hydrate in 3.5 mL of 0.5M sulfuric acid and 10 mL
of water. Add 100 mL of picric acid solution R1, heat on a
Phthalic Anhydride Isobenzofuran-1 ,3-dione;
water bath for 15 minutes, cool and filter. Wash the
C SH 4 0 3 = 148.1 (85-44-9)
precipitate with water until the washings are free from sulfate.
General reagent grade of commerce containing not less than Shake the precipitate with water to produce a saturated
99.0% of C SH 4 0 3. solution and filter.
0
Melting point, 130 to 132°. Piperazine Hydrate Of the British Pharmacopoeia.
Phthalic Anhydride Solution Dissolve 42 g of phthalic Piperidine Hexahydropyridine; CSH11N = 85.2 (110-89-
anhydride in 300 mL of anhydrous pyridine. Allow to stand for 4)
16 hours.
Store protected from light and use within 1 week.
Picein 1-[4-(~-D-Glucopyranosyloxy)phenyl] ethanone;
Piperine (2E,4E)-I-(piperidin-I-yl)-5-(l,3-benzodioxol-5-
Cl~IS07 = 298.3· (530-14-3)
yl)penta-2,4-dien-1-one, l-Piperoyl-piperidine, 1-[(2E,4E)-5-
General reagent grade of commerce. (3,4-Methylenedioxyphenyl)-l -oxo-2,4-
Melting point, 194° to 195°. pentadienyl]piperidine; C 17H I9N 0 3 = 285.3 (94-62-2)
Picric Acid 2,4,6-Trinitrophenol; Piperitone 6-Isopropyl-3-methyl-cyclohex-2-en-l-one;
CóH3N307 = 229.1 (88-89-1) ClOH1óO = 152.2 (89-81-6)
Analytical reagent grade of commerce. Pirimiphos-ethyl C13H2~3 0 3 PS = 333.4 (23505-41-1 )
Yellow prisms or plates moistened with an equal weight of Use a grade of commerce suitable for pesticide residue
water for safety; explodes when heated rapidly or subjected to analysis. A suitable certified reference solution (lO nglflL in
percussion. eyclohexane) may be used.
Store moistened with water. Melting point, 15° to 18°.
Picric Acid Solution A 1% w/v solution of picric acid. Plasma, Citrated Rabbit Colleet blood by intracardiae
Picric Acid Solution Rl Add 0.25 mL of 10M sodium puneture from a rabbit that has been fasted for 12 hours
hydroxide to 100 mL of a saturated solution of picric acid in prior to the eollection, using a plastic syringe with a No. 1
water. needle containing a suitable volume of a 3.8% w/v solution
Picrolonic Acid 3-Methyl-4-nitro-1-(4-nitrophenyl)-5- of sodium citrate so that the final volume ratio of eitrate
pyrazolone; ClOHsN4 0 S = 264.2 (550-74-3) solution to blood is 1:9. Separate the plasma by
centrifugation at 1500 to 1800 g at 15° to 20° for
30 minutes.
A yellow or brownish yellow, crystalline powder; melting
Store at 0° to 6° and use within 4 hours of collection.
point, about 116°.
Plasma, Platelet-poor Withdraw 45 mL of human blood
into a 50 mL plastic syringe eontaining 5 mL of a sterile
Sensitivity Dissolve 25 mg in 10 mL of warm water 3.8% w/v solution of sodium citrate. Immediately centrifuge
containing 0.1 mL of glacial acetic acid; to 1 mL of this the citrated whole blood at 1150 g for 30 minutes at 4°.
solution add 1 mL of a 0.05% w/v solution of calcium Remove the upper two thirds of the supernatant plasma
chlmide previously heated to 60°. A bulky precipitate is using a plastie syringe and immediately centrifuge at 3500 g
produced within 5 minutes. for 30 minutes at 4°. Remove the upper two thirds of the
liquid and freeze it rapidly in suitable quantities in plastic
2014 Appendix 1 A V-AI03
tubes at a temperature of -40' or below. Use plastic or swirling until thawing is complete; once thawed it should be
siliconised equipment throughout. kept at 10° to 20° and used without delay. The thawed
Plasma Substrate Substrate plasma substrate plasma may be lightly centrifuged if necessary;
Separate the plasma from 9 volumes of human or bovine filtration procedures should not be used.
blood collected in 1 volume of a 3.8% w/v solution of sodium Plasma Substrate R2 Prepare from human blood
citrate or from 3.5 volumes of human or bovine blood containing less than 1% of the normal amount of factor IX.
collected in 1 volume of a solution containing 2% w/v of Collect 9 volumes of the blood into 1 volume of a 3.8% w/v
disodium hydrogen citrate and 2.5% w/v of D-glucose. In the solution of sodium citrate. Store in small amounts in plastic
0
former case, prepare the substrate plasma on the day of tubes at a temperature of - 30 or below.
collection of the blood. In the latter case, the substrate Plasma Substrate R3 Prepare from human blood
plasma may be prepared up to 2 days after collection of the containing less than 1% of the normal amount of factor XI.
blood. Collect the blood into one-ninth its volume of a 38 gIL
Store at - 20°. solution of sodium citrate.
Plasma Substrate Deficient in Factor V Preferably use Store in small amounts in plastic tubes at a temperature
congenitally deficient plasma or, altematively, prepare in the of - 30° or lower.
following manner. Separate the plasma from human blood Plasminogen, Human (9001-91-6)
collected in 0.1 of its volume of a 1.34% w/v solution of A substance present in blood which may be activated to
sodium oxalate and incubate at 37° for 24 to 36 hours. This plasmin, an enzyme that Iyses fibrin in blood dots .
plasma should have a clotting time, when tested by the assay Platelet Substitute To 0.5 to 1 g of phospholipid add
method given under coagulation factor V solution, of 70 to 20 mL of acetone and allow to stand for 2 hours with
100 seconds; if the clotting time is les s than 70 seconds, frequent shaking. Centrifuge for 2 minutes and discard the
incubate the plasma for a further 12 to 24 hours. supernatant liquid. Dry the residue using a water pump, mix
0
Store, in small amounts, at _20 or below. with 20 mL of chloroform and shake for 2 hours. Filter under
Plasma Substrate Rl Substrate plasma Rl vacuum and suspend the residue obtained in 5 to 10 mL of
Use water-repellent equipment (made from materials such as satine solution.
suitable plastics or suitably siliconised glass) for taking and Prepare a dilution in saline solution so that it will give clotting
handling the blood. Collect a suitable volume of blood from time differences between consecutive dilutions of the
each of an appropriate number of sheep. (A volume of reference preparation used in the Assay of factor IX fraction
285 mL of blood added to 15 mL of anticoagulant solution of about 10 seconds.
is considered suitable; smaller volumes may be collected. Store the dilute suspensions at - 30 0 and use within 6 weeks.
Whatever volume is collected it is considered advisable to use Plutonium-242 Spiking Solution
at least 5 sheep.) Take the blood, either from a live animal or
immediately after slaughter, using a needle attached 10 a Contains 50 Bq/L 242Pu and a 134 gIL solution of
suitable cannula which is long enough 10 reach the bottom of lanthanwn chloride heptahydrate in a 284 gIL solution of nitric
the collecting vessel. Discarding the first few millilitres and acid.
collecting only free-flowing blood, collect the blood into Poloxamer 124
sufficient of an anticoagulant solution containing 8.7 g of General reagent grade of commerce.
sodium citrate and 4 mg of aprotinin in 100 mL of water to Poly[ (cyanopropyl)methylphenylmethylsiloxane]
give a final ratio of blood to anticoagulant solution of 19: 1. Gas chromatographic reagent grade of commerce with an
During and immediately after collection swirl the flask gently
average molecular weight of 8000 containing 25% of
to ensure mixing, but do not allow frothing 10 occur. When cyanopropyl groups, 25% of phenyl groups and 50 % of
collection is complete, close the flask, cool to 10" to 15° and methyl groups.
then pool the contents of all the flasks with the exception of
any that show obvious haemolysis or clots and keep the Poly[ (cyanopropyl) (methyl)][ (phenyl) (methyl) ]siloxane
pooled blood at 10° to 15°. See Poly[(cyanopropyl) methylphenylmethylsiloxanej.
As soon as possible and, in any case, within 4 hours of Poly[ (cyanopropyl) (phenyl)][ (dimethyl]siloxane
collection, centrifuge the pooled blood at 1000 to 2000 g at Gas chromatographic reagent grade of commerce containing
100 to 15° for 30 minutes. Separate the resulting supematant 6% of (cyanopropyl) (phenyl) groups and 94% of dimethyl
liquid and centrifuge it at 5000 g for 30 minutes. (More groups.
powerful centrifugation, for example at 20,000 g for Poly( cyanopropylphenyl) (14) (methyl) (86) siloxane
30 minutes, may be used if necessary 10 clarifY the plasma at Stationary phase for chromatography.
this stage but filtration procedures should not be used.)
Contains 14% of cyanopropylphenyl groups and 86% of
Separate the resulting supernatant liquid and, without delay,
methyl groups.
mix thoroughly and distribute the resulting substrate plasma
into small stoppered containers in portions suffic ient for a Poly( cyanopropyl) (phenylmethyl)siloxane
complete heparin assay (for example, 10 to 30 mL). Without Gas chromatographic reagent grade of commerce containing
delay, freeze rapidly at a temperature below - 70° (for 90% cyanopropyl and 10% phenylmethyl groups.
example, by plunging the containers into liquid nitrogen) and Poly( cyanopropyl) (7) (phenyl) (7)methyl) (86)siloxane
store at a temperature below - 30°. Gas chromatographic reagent grade of commerce.
The prepared plasma is considered suitable for use as Polysiloxane substituted with 7% of cyanopropyl groups, 7%
substrate plasma in the assay for heparin if, under the of phenyl groups and 86% of dimethyl groups.
conditions of the assay, it gives a clotting time appropriate to
Poly( cyanopropyl) siloxane Polysiloxane substituted with
the method of detection used and if it provides reproducible,
100% of cyanopropyl groups.
steep log dose-response curves. When required, thaw a
portion of substrate plasma in a water bath at 37°, gently Gas chromatographic grade of commerce.
Poly( dirnethyl) (diphenyl) (divinyl)siloxane Polyethylene Glycol 20,000 Macrogol 20,000

Chromatographic reagent grade of commerce containing Chromatographic reagent grade of commerce.
94% of methyL groups, 5% of phenyl groups and 1% of vinyl A hard, white, waxy solidó soluble in water with gel
groups. SE54 formation.
Poly(dimethyl) (diphenyl)siloxane Polyethylene Glycol 20,000 2-Nitroterephthalate
Chromatographic reagent grade of commerce containing General reagent grade of commerce.
95% of methyl groups and 5% of phenyl groups.
A white or almost white, hard, waxy solid.
Poly( dimethyl) (diphenyl)siloxane, base-deactivated Polyethylene Glycol Adipate Macrogol adipate
Base-deactivated stationary phase for gas chromatography
specially designed for amine analysis. Gas chromatographic reagent grade of commerce.
Contains 95% of methyl groups and 5% of phenyl groups. A white, waxy mass; melting point, about 43°.
DB-5, SE52 Polyethylene Glycol Succinate Macrogol succinate
Poly( dimethyl) (75) (diphenyl) (25)siloxane Gas chromatographic reagent grade of commerce.
Chromatographic reagent grade of commerce containing A white, crystalline powder; melting point, about 102°.
75% of methyl groups and 25% of phenyl groups. Polyethyleneglycol, Polar-deactivated Stationary phase
Poly(dimethyl)siloxane Silicone gum rubber (methyl) for gas chromatography.
Gas chromatographic reagent grade of commerce. Polymethacrylate Gel
Organosilicon polymer with the appearance of a semi-liquid, A methacrylate-based size-exclusion stationary phase for
colourless gum. The infrared absorption spectrnm, water-soluble samples.
Appendix JI A, obtained by applying the reagent, if necessary Polymethacrylate Gel, Hydroxylated
dispersed in a few drops of carbon tetrachloride, to a sodium Gel based on hydroxylated methacrylic acid polymer.
chloride plate do es not show absorption at 3053 cm- l, Stationary phase for size-exclusion chromatography.
corresponding to vinyl groups.
Polymethylphenylsiloxane Average molecular weight,
Poly( dimethyl) (85) (diphenyl) (15)siloxane 4000.
Stationary phase for chromatography. Chroma1Ographic grade of commerce containing about 50%
Contains 85% of methyl groups and 15% of phenyl groups; of methyl groups and 50% of phenyl groups.
PS086. A very viscous liquid (about 1300 mPa·s); weight per mL,
Polyether Hydroxylated Gel for Chromatography about 1.09 g.
Chromatographic reagent grade of commerce. Poly[methyl(95)phenyl(5) ]siloxane Polysiloxane
Gel with a small particle size having a hydrophilic surface containing 95% of methyl groups and 5% of phenyl groups.
with hydroxyl groups. It has an exclusion limit for dextran of Chroma1Ographic reagent grade of commerce.
molecular weight 2 x 10 5 to 2.5 X 10 6 . Poly[ methyl(94)phenyl(5)vinyl(l) ]siloxane Polysiloxane
Polyethylene Glycol 200 Macrogol 200; (25322-68-3) containing 94% of methyl groups, 5% of phenyl groups and
General reagent grade of commerce. 1% of vinyl groups.
A viscous liquidó d~g, about 1.127; nbo, about 1.450. Chromatographic reagent grade of commerce.
Polyethylene Glycol 200 Rl Macrogol 200 R1 Poly[ methyl(trifluoropropyImethyl) siloxane ]
Place 500 mL of polyethylene glycol 200 in a 1000-mL round- Chroma1Ographic reagent grade of commerce containing
bottomed fiask. Using a rotary evaporator remove any volatile 50% of trifiuoropropylmethyl groups and 50% of methyl
components by applying a temperature of 60° and a pressure groups.
of 1.5 to 2.5 kPa for 6 hours . Polyoxyethylated Castor Oil
Po1yethylene Glycol 300 Macrogol 300; (25322-68-3) General reagent grade of commerce.
General reagent grade of commerce. A light yellow liquid which becomes clear aboye 26°.
A viscous liquidó weight permL, about 1.13 g; n~, about Polyoxyethylene 23 Lauryl Ether Brij 35;
1.465; viscosity at 25°, about 80 mPa·s, Appendix V H, CSSHl20024 = 1199.6 (9002-92-0)
Method JI. General reagent grade of commerce.
Polyethylene Glycol 400 Macrogol 400; (25322-68-3) Melting point, about 43°; boiling point, about 100°.
General reagent grade of commerce. Polysorbate 20 Of the British Pharmacopoeia.
A viscous liquidó weight per mI.., about 1.13 g; freezing Polysorbate 80 Of the British Pharmacopoeia .
point, about 6°; viscosity, about 130 mPa·s, Appendix V H,
Polystyrene 900-1000 (9003-53-6)
Method Il.
Organic standard used for calibration in gas chromatography.
Polyethylene Glycoll000 Macrogol 1000; (25322-68-3)
Molecular weight, about 950.
Potassium Acetate Of the British Pharmacopoeia .
A white, waxy mass; freezing point, about 35°; viscosity,
about 25 mPa·s for a 50% w/w solution, Appendix V H, Potassium Antimonate(v) Potassium pyroantimonate;
Method II. KSb0 3,3H 20 = 262.9 (12208-13-8)
Polyethylene Glycol1500 Macrogol 1500; (25322-68-3) Analytical reagent grade of commerce.
General reagent grade of commerce. Potassium Antimonate(v) Solution Potassium
pyroantimonate solution
A white, waxy mass; freezing point, 43° 10 48°; viscosity,
about 70 mPa·s for a 50% w/w solution, Appendix V H , Dissolve 2 g of potassium antimonate(v) in 95 mL of hot
Method JI. water, cool rapidly and add a solution containing 2.5 g of
2014 Appendix 1 A V-A10S
potassium hydroxlde in 50 mL of water and 1 mL of 2M sodium sulfide solution. No colour is evolved even after addition of
hydroxide. Allow to stand for 24 hours, filter and dilute to 5 mL of dilure hydrochloric acid.
150 mL with water. Potassium Cyanide Solution PbT Dissolve 10 g of
Potassium Bicarbonate See Potassium hydrogen carbonate. potassium cyanide in 90 mL of water, add 2 mL of hydrogen
Potassium Bicarbonate Soludon, Saturated Methanolic peroxlde solution (20 vo!), allow to stand for 24 hours, dilute
See Saturated methanolic potassium hydrogen carbonate solution. to 100 mL with water and filter.
Potassium Borohydride Potassium tetrahydroborate; Potassium Dichromate Dipotassium dichromate;
KBH4 = 53.94 (13762-51-1) K2Cr207 = 294.2 (7778-50-9)
General reagent grade of commerce. Analytical reagent grade of commerce.
Potassium Bromate KBr03 = 167.0 (7758-01-2) Potassium dichromate used for the calibration of
spectrophotometers contains not less than 99.9% of
KZCr20 7, ca1culated with reference to the substance dried at
White, granular powder or crystals. 130°.
Potassium Bromide Of the British Pharmacopoeia. Assay Dissolve 1 g in sufficient water to produce 250 mL.
Potassium bromide used for infrared absorption spectrophotometry Add 50 mL of this solution to a freshly prepared solution of
also complies with the following additional test. 4 g of potassium iodide, 2 g of sodiwn hydrogen carbonate and
The infrared absorption spectrum, Appendix II A, of a disc 6 mL of hydrochloric acid in 100 mL of water in a 500-mL
2 mm thick prepared from material previously dried at 250 0
fiask. Stopper the fiask and allow to stand protected from
for 1 hour has a substantially fiat baseline over the range light for 5 minutes. Titrate with O.lM sodiu111 thiosulfate VS
4000 to 620 cm- Ji it exhibits no maxima with an absorbance using 1 mL of iodide-free starch solution as indicator. Each mL
greater than 0.02 aboye the baseline with the exception of of O.IM sodium thiosulfate VS is equivalent to 4.903 mg of
maxima due to water at 3440 and 1630 cm- J. K 2 Cr2 0 7.
Potassium Carbonate Anhydrous potassium carbonate; Potassium Dichromate Solution A 10.6% w/v solution
K 2 C0 3 = 138.2 (584-08-7) of POtasSiU111 dichromate.
Analytical reagent grade of commerce. Potassium Dichromate Solution, Dilute A 7.0% w/v
A white, granular, hygroscopic powder. solution of potassium dichromate.
Store in an airtight container. Potassium Dichromate Solution Rl A 0.5 % w/v
solution of POtasSiU111 dichr0111ate.
Potassium Carbonate Sesquihydrate
K zC0 3, 1'/2 H 20 = 165.2 (6381-79-9) Potassium Dihydrogen Citrate C 6H 7K0 7 = 230.2
Potassium Chlorate KCI0 3 = 122.6 (3811-04-9) Potassium Dihydrogen Orthophosphate See Potassiu111
dihydrogen phosphate
Potassium Dihydrogen Phosphate Of the British
A white powder, granules or crystals.
Pharmacopoeia.
Potassium Chloride Of the British Pharmacopoeia.
Potassium Dihydrogen Phosphate, O.2M A solution of
Potassiwn chloride used for infrared absorption spectrophotometly potassiwn dihydrogen orthophosphate containing 27.22 g per
also complies with the following additional test. litre of KH 2P0 4.
The infrared absorption spectru111, Appendix II A, of a disc Potassium Ferricyanide See Potassium
0
2 mm thick prepared from material previously dried at 250 hexacyanoferrate (m) .
for 1 hour has a substantially fiat baseline over the range
Potassium Ferricyanide Solution See POtaSSiU111
4000 to 620 cm- Ji it exhibits no maxima with an absorbance
hexacyanoferrate(m) solution.
greater than 0.02 aboye the baseline with the exception of
maxima due to water at 3440 and 1630 cm- J. Potassium Ferriperiodate Soludon Dissolve 1 g of
POtasSiU111 periodate in 5 mL of a freshly prepared 12% w/v
Potassium Chloride, O.IM A solution of POtasSiU111 chlon"de
solution of potassium hydroxide, add 20 mL of water and
containing 7.46 g of KCI in 1000 mL.
1.5 mL of iron(m) chloride solurion R1 and add sufficient of a
Potassium Chromate K2Cr04 = 194.2 (7789-00-6) freshly prepared 12% w/v solution of potassium hydroxide to
Analytical reagent grade of commerce. produce 50 mL.
Yellow crystals. Potassium Ferrocyanide See Potassiwn
Potassium Chromate Soludon A 5% w/v solution of hexacyanoferrate(lI) .
potassiu111 chr0111ate. Potassium Ferrocyanide Soludon See POtaSSiU111
Produces a red precipitate with silver nitrate in neutral hexacyanofen°ate(n) solution.
solutions. Potassium Fluoride KF = 58.1 (7789-23-3)
Potassium Citrate Of the British Pharmacopoeia. General reagent grade of commerce.
Potassium Cyanide KCN = 65.12 (151-50-8) Colourless crystals or white crystalline powder.
Analytical reagent grade of commerce. Potassium Hexacyanoferrate(u) Potassium ferrocyanide;
A white, crystalline powder or white mass or crystals. I(,¡Fe(CN)6,3H 20 = 422.4 (14459-95-1)
Potassium Cyanide Soludon A 10% w/v solution of Analytical reagent grade of commerce.
POtasSiU111 cyanide. Transparent, yellow crystals.
Potassium Cyanide Solution, Lead-free Potassium Hexacyanoferrate(ru) Potassium ferricyanide;
K3Fe(CN) 6 = 329.3 (13746-66-2)
The solution complies with the following test: take 10 mL of
the solution, add 10 mL of water and 10 mL of hydrogen Analytical reagent grade of commerce.
Red crystals.
Potassium Hexacyanoferrate(n) Solution A 5.3 % w/v Potassium Hydroxide, Methanolic Solutions of the
solution of potassium hexacyanoferrate(II). requisite molarity may be obtained by dissolving the
Potassium Hexacyanoferrate(m) Solution Wash 5 g of appropriate amount of potassium hydroxide in sufficient
potassium hexacyanoferrate(m) crystals with a little water and methanol 10 produce 1000 mL.
dissolve in sufficient water to produce 100 mL. Potassium Hydroxide Solution, Alcoholic Dissolve 3 g
Prepare irnmediately before use. of potassium hydroxide in 5 mL of water and dilute 10 100 mL
with aldehyde-free ethanol (96%) and decant the c1ear
Potassium Hexacyanoferrate(m) Solution, Dilute
solution. The solution should be almost colourless.
Wash about 1 g of potassium hexacyanoferrate(m) crystals with
a little water and dissolve the washed crystals in 100 mL of Potassium Hydroxide Solution R1, Alcoholic Dissolve
water. 6.6 g of potassium hydroxide in 50 mL of water and dilute to
1000 mL with absolute ethanol.
Produces a blue colour with solutions of iron(n) salts.
Potassium Iodate KI0 3 = 214.0 (7758-05-6)
Prepare immediately before use .
Potassium Hyaluronate (31799-91-4)
A white, crystalline powder.
General reagent grade of commerce obtained from human
umbilical cords and freeze dried. Potassium Iodide Of the British Pharmacopoeia.
Protein, not more than 2%; chondroitin sulfate, not more Potassium Iodide and Starch Solution Dissolve 0.75 g
than 3%. of potassium iodide in 100 mL of water, heat 10 boiling and
add, whilst stirring, a solution of 0.5 g of soluble starch in
Potassium Hyaluronate Stock Solution A 0.05% w/v
35 mL of water. Boil for 2 minutes and allow 10 coo!.
solution of potassium hyaluronate.
S10re below 0° and use within 30 days.
Sensitivity to iodine To 15 mL of the reagent add
Potassium Hydrogen Carbonate Potassium bicarbonate;
0.05 mL of glacial acetic acid and 0.3 mL of 0.0005M iodine
KHC0 3 = 100.1 (298-14-6)
VS. A blue colour is produced.
Potassium Iodide Solution A 16.6% w/v solution of
Potassium Hydrogen Carbonate Solution, Saturated potassium iodide.
Methanolic Dissolve 0.1 g of potassium hydrogen carbonate
Potassium Iodide Solution, Dilute A 10% w/v solution
in 0.4 mL of water by heating on a water bath. Add 25 mL
of potassium iodide.
of methanol and swirl, keeping the solution on the water bath
until dissolution is complete. Potassium Iodide Solution, Iodinated Dissolve 2 g of
iodine and 4 g of potassium iodide in 10 mL of water. When
Use a freshly prepared solution.
solution is complete, add sufficient water to produce
Potassium Hydrogen Phthalate 100 mL.
C sH sK0 4 = 204.2 (877-24-7)
Potassium Iodide Solution, Iodinated R1
Dissolve 500 mg of iodine and 1.5 g of potassium iodide in
Potassium Hydrogen Phthalate, 0.2M A solution of water, dilute to 25 mL with water.
potassium hydrogen phthalate containing 40.84 g of C sH sK0 4
Potassium Iodide Solution, Saturated A saturated
in 1000 mL.
solution of potassium iodide in carbon dioxide-free water; it
Potassium Hydrogen Sulfate Potassium bisulfate; contains sorne undissolved crystals.
potassium bisulphate; potassium hydrogen sulphate;
Store protected from Iight and discard if it fails 10 comply
KHS0 4 = 136.2 (7646-93-7)
Add 0.1 mL of starch solution 10 0.5 mL of the reagent in
Colourless, transparent, hygroscopic crystals. 30 mL of a mixture of 3 volumes of 6M acetic acid and
S10re in an airtight container. 2 volumes of chloroform. lf a blue colour is produced it is
Potassium Hydrogen Tartrate See Potassium hydrogen discharged on the addition of not more than 0.05 mL of
(+) -tartrate. O.lM sodium thiosulfate VS.
Potassium Hydrogen (+)-Tartrate Potassium Iodobismuthate Solution Dissolve 8 g of
C 4 H sK0 6 = 188.2 (868-14-4) potassium iodide in sufficient water to produce 20 mL and add
Analytical reagent grade of commerce. the solution 10 a mixture of 0.85 g of bismuth oxynitrate,
40 mL of water and 10 mL of glacial acetic acid.
A white, crystalline powder or colourless, slightly opaque
crystals. Potassium Iodobismuthate Solution, Acid
Potassium Hydroxide Of the British Pharmacopoeia. Dissolve 1.7 g of bismuth oxynitrate in a mixture of 80 mL of
water and 20 mL of glacial acetic acid, warming if necessary,
Potassium Hydroxide, 2M Alcoholic Dissolve 12 g of
cool, add 100 mL of a 50% w/v solution of potassium iodide
potassium hydroxide in 10 mL of water and add sufficient
and mix. Dilute 10 mL to 100 mL with water, add 10 mL of
ethanol (96%) 10 produce 100 mL.
glacial acetic acid, mix, add 0.12 g of iodine and shake until
Potassium Hydr~xide, Ethanolic Solutions of the the iodine has completely dissolved.
requisite molarity may be obtained by dissolving the
Store at a temperature of 2° to 8° and use within 2 weeks.
appropriate amount of potassium hydroxide in sufficient
ethanol (96%) to produce 1000 mL. Potassium Iodobismuthate Solution, Dilute Dissolve
100 g of (+)-tartaric acid in 500 mL of water and add 50 mL
Potassium Hydroxíde in Alcohol (10% v/v), 0.5M
of potassium iodobismuthate solution R 1.
Dissolve 28 g of potassium hydroxide in 100 mL of ethanol
(96%) and add sufficient water to produce 1000 mL. Potassium Iodobismuthate Solution R1 Dissolve 100 g
of (+)-tartaric acid in 400 mL of water and add 8.5 g of
bismuth oxynitrate. Shake for 1 hour, add 200 mL of a
2014 Appendíx 1 A V-A107
40% w/v solution of potassium iodide and shake well. Allow to Aqueous solutions decompose at room temperature and
stand for 24 hours and filter. more rapidly on warming.
Store protected from light. Store in a cool place.
Potassium Iodobismuthate Solution R2 Suspend 1.7 g Potassium Plumbite Solution Dissolve 1.7 g of lead
of bismuth oxynitrate and 20 g of (+)-tartane acid in 40 mL aeetate, 3.4 g of potassium eitrate and 50 g of potassium
of water. To the suspension add 40 mL of a 40% w/v hydroxide in sufficient water to produce 100 mL.
solution of potassium iodide, stir for 1 hour and filter. This Potassium Pyroantimonate See Potassium antimonate(v).
stock solution may be kept for several days protected from Potassium Pyroantimonate Solution See Potassium
light. Immediately before use, mix 5 mL of the stock antimonate(v) solution.
solution with 15 mL of water.
Potassium Sodium (+)-Tartrate Sodium potassium (+)-
Potassium Iodobismuthate Solution R3 Dissolve 0.17 g tartrate; C 4 H 4 KNaO ó,4H 20 = 282.2 (6381-59-5)
of bismuth subnitrate in a mixture of 2 mL of glacial aeeLÍe acid
and 18 mL of water. Add 4 g of potassium iodide, 1 g of Analytical reagent grade of commerce.
iodine and dilute to 100 mL with 1M su/fun'e aeid. Potassium Sorbate Sorbic acid potassium salt;
Potassium Iodobismuthate Solution R4 C óH 7 K0 2 = 150.2 (110-44-1)
Dissolve 1.7 g of bismuth subnitrate in 20 mL of glacial aeeLÍe General reagent grade of commerce.
aeid. Add 80 mL of disLÍlled water, 100 mL of a 40.0% w/v Potassium Sulfate Dipotassium sulfate, dipotassium
solution of potassium iodide, 200 mL of glacial aeeLÍe aeid and sulphate, potassium sulphate; K 2S0 4 = 174.3 (7778-80-5)
dilute to 1000 mL with disLÍlled water. Mix 2 volumes of this Analytical reagent grade of commerce.
solution with 1 volume of a 20.0% w/v solution of banum Potassium Tartrate See Dipotassium (+)-tartrate.
ehlonde.
Potassium Tetraiodomercurate Solution Dissolve
Potassium Iodobismuthate Solution R5 1.35 g of mereury(Il) ehlonde in 50 mL of water, add 5 g of
To 0.85 g of bismuth subnitrate add 10 mL of glacial aeetie potassium iodide, mix and add sufficient water to produce
acid and gently heat until completely dissolved. Add 40 mL 100 mL.
of water and allow to cool. To 5 mL of this solution, add Potassium Tetraiodomercurate Solution, Alkaline
5 mL of a 400 gIL solution of potassium iodide, 20 mL of Dissolve 11 g of potassium iodide and 15 g of mereury(n)
glacial aeeLÍe acid and 70 mL of water. iodide in water and dilute to 100 mL with water. Irnmediately
Potassium Iodop1atinate Solution Add 5 mL of a before use, mix the solution with an equal volume of a
5% w/v solution of chloroplaLÍnie(IV) acid to 45 mL of dilute 25% w/v solution of sodium hydroxide.
potassium iodide soluLÍon and dilute to 100 mL with water. Potassium Tetroxalate . Potassium trihydrogen dioxalate;
Store in an amber glass container. C 4 H 3KO s,2H 20 = 254.2 (6100-20-5)
Potassium Mercuri-iodide Solution, Alkaline To 3.5 g Analytical reagent grade of commerce.
of potassium iodide add 1.25 g of mercury(Il) ehlonde dissolved A white, crystalline powder.
in 80 mL of water and a cold, saturated solution of Potassium Thiocyanate KCNS = 97.18 (333-20-0)
mereury(Il) ehlonde in water, stirring continuously, until a
slight red precipitate remains. Dissolve 12 g of sodium Analytical reagent grade of commerce.
hydroxide in the resulting solution and add a little more of the Colourless crystals; deliquescent.
saturated solution of mercury(n) chloride and sufficient water Store in an airtight container.
to produce 100 mL. Allow to stand and decant the clear, Potassium Thiocyanate Solution A 9.7% w/v solution of
supernatant liquid. potassium thiocyanate.
Potassium Nitrate KN0 3 = 101.1 (7757-79-1) Povidone Of the British Pharmacopoeia.
Analytical reagent grade of commerce. Prednisolone C21H2S0S = 360.5 (50-24-8)
Colourless crystals. General reagent grade of commerce.
Potassium Periodate Potassium metaperiodate; Hygroscopic crystalline powder; melting poim, about 230°,
KI0 4 = 230.0 (7790-21-8) with decomposition; [al~, about +97 (1 % w/v in
Analytical reagent grade of commerce. 1,4-dioxan).
A white, crystalline powder or colourless crystals. Prednisolone 21-Acetate C23H300 ó = 402.5 (52-21-1)
Potassium Pennanganate Of the British Pharmacopoeia. General reagent grade of commerce.
Potassium Pennanganate and Phosphoric Acid Procaine Hydroch1oride Of the British Pharmacopoeia.
Solution Dissolve 3 g of potassium permanganate in a Proline Of the British Pharmacopoeia.
mixture of 15 mL of orthophosphone acid and 70 mL of water o-Prolyl-L-phenylalanyl-L-arginine 4-Nitroanilide
and add sufficient water to produce 100 mL.
Hydroch1oride C2óH 3óCI2NsOs = 612 (62354-56-7)
Potassium Pennanganate Solution A 3% w/v solution
of potassium permanganate.
Propane-l,2-diol See Propylene glycol
Potassium Pennanganate Solution, Dilute A 1.0% w/v
solution of potassium permanganate. Propane-l,3-diol 1,3-Dihydroxypropane;
C 3H s0 2 = 76.1 (504-63-2)
Potassium Perrhenate KRe04 = 289.3 (10466-65-6)
Colourless, viscous liquido
Boiling point, about 214°; melting point about -27°.
Potassium Persulfate Dipotassium peroxodisulfate,
dipotassium peroxodisulphate, potassium persulphate; Propanol See Propan-l-ol.
K 2S20 S = 270.3 (7727-21-1) Propanol Rl Propanol of the British Pharmacopoeia.
General reagent grade of commerce. 2-Propanol See Propan-2-ol.
V-A10S Appendix 1 A 2014
2-Propanol Rl See Propan-2-ol R1 . Propylene Oxide C 3H 60 = 58.1 (75-56-9)

2-Propanol R2 Isopropyl Alcohol of the British General reagent grade of commerce.
Pharmacopoeia. Boiling point, about 34°; n~o, about 1.366.
Propan-l-ol Propanol; n-propyl alcohol; Protamine Sulfate Of the British Pharmacopoeia.
C 3H sO = 60.10 (71-23-8) Protopine Hydrochloride 5-Methyl-4,6,7,14-
Analytical reagent grade of commerce. tetrahydrobis [1 ,3]benzodioxolo [4,5-c:5' ,6'-g] azecin-13 (5H)-
A colourless liquid; boiling point, about 97°; dig, 0.802 to one hydrochloride; C2oH20CINOs = 389.8 (6164-47-2)
0.806 . Not les s than 95 % distils between 96° and 99°. Pteroic Acid 4-[[ (2-Amino-4-oxo-1,4-dihydropteridin-6-
Propan-2-o1 Isopropyl alcohol; 2-propanol; yl)methyl]amino]benzoic acid;
C 3H sO = 60.10 (67-63-0) C14H1 2N603 = 312.3 (119-24-4)
Analytical reagent grade of commerce. Crystals, soluble in solurions of alkali hydroxides.
A colourless liquid; boiling point, 81 ° to 83°; dig, about Puerarin 7,4 '-Dihydroxy-8-C-glucosyliso-haloprone; 8-~
0.785. D-Glucopyranosyl-7 -hydroxy-3-(4-hydroxyphenyl)-4H-1-
Propan-2-o1 Rl Propan-2-ol complying with the following benzopyran-4-one; C21 H 200 9 = 416.4 (3681-99-0)
tests. General reagent grade of commerce.
Refractive index About 1.378. Pulegone (+ )-p-Menth-4-en-3-one; (R)-2-isopropylidene-
Transmittance Not less than 25% at 210 nm, 55% at 5-methylcyclohexanone; C IOH 16 0 = 152.2 (89-82-7)
220 nm, 75 % at 230 nm, 95% at 250 nm and 98% at General reagent grade of commerce.
260 nm, determined using water in the reference cel!. df~, about 0.937; n~, about 1.485 to 1.489; [a]~o, +19.5 to
Water Not more than 0.05% w/w, Appendix IX C. Use + 22.5; boiling point, 222 0 10 224°.
10 g. Pulegone used in gas chromatography complies with the following
Propanolamine See 3-Aminopropanol. test.
Propetamphos C lO H 20N0 4PS = 281.3 (31218-83-4) Assay Examine by gas chroma1Ography under the
Use a grade of commerce suitable for pesticide residue conditions described in the test for Chromatographic profile
analysis. A suitable certified reference solution (10 ng/IlL in in the monograph for Peppermint Oi! using the reagent
cyclohexane) may be used. being examined.
Propidium Iodide 3,8-Diamino-5- The area of the principal peak is not less than 98.0% of the
[3 (diethylmethylammonio) propyl]-6-phenylphenanthridinium total area of the peaks.
diiodide; C27H34I2N4 = 668.4 (25535-16-4) Pumice Powder Pumice of commerce, powdered and
Dark red solido sifted, which passes through a 710 11m sieve, but is retained
Propionaldehyde Propanal; C 3H 60 = 58.1 (123-38-6) on a 250 11m sieve.
General reagent grade of commerce. Putrescine 1,4-Butanediamine; tetramethylenediamine;
C 4H 12N 2 = 88.15 (110-60-1)
Melting point, about - 81 °; boiling point, about 49°; n~o,
about 1.365; dig, about 0.81.
Propionic Acid C 3H 60 2 = 74.1 (79-09-4) Boiling point, about 159°; melting point, about 23°.
General reagent grade of commerce. Pyrazine-2-carbonitrile 2-Cyanopyrazine;
C SH 3N 3 = 105.1 (19847-12-2)
di g, about 0.993; n~, about 1.387; boiling point, about
141 °. Appearance, clear, pale yellow liquido
Propionic Anhydride C 6H lO 0 3 = 130.1 (123-62-6) Content, minimum 99%.
General reagent grade of commerce. Boliling point, about 199°.
A colourless liquid; weight per mL, about 1.01 g; boiling Pyridin-2-amine 2-Arninopyridine;
point, about 167°. C SH 6N 2 = 94.1 (504-29-0)
A clear, colourless liquido Large crystals soluble in water and in ethanol (96 %).
Propionic Anhydride Reagent Dissolve 1 g of toluene-p- Boiling point, about 210°; melting point, about 58°.
sulfonic acid in 30 mL of glacial acetic acid, add 5 mL of Pyridine CsHsN = 79.10 (110-86-1)
propionic anhydride and allow to stand for at least 15 minutes. Analytical reagent grade of commerce.
Use within 24 hours of preparation. A colourless liquid; boiling point, about 115°.
Propranolol Hydrochloride Store in an airtight container.
C 16H 21NOz,HCI = 295 .8 (318-98-9) Pyridine, Anhydrous
General reagent grade of commerce. Dry pyridine over anhydrous sodium carbonate, filter and disti!.
Melting point, about 164°. Complies with the following test.
Propyl Acetate C S H lO 0 2 = 102.1 (109-60-4) Water Not more than 0.01 % w/w, Appendix IX C.
General reagent grade of commerce. Pyridine Bromide Solution Dissolve 8 g of pyridine and
Boiling point, about 102°; dig, about 0.888. 5.4 mL of sulfuric acid in 20 mL of glacial acetic acid, keeping
Propyl4-Hydroxybenzoate See Propyl the mixture coo!. Add 2.6 mL of bromine dissolved in 20 mL
parahydroxybenzoate of glacial acetic acid and di!ute to 1000 mL with glacial acetic
PropyI Parahydroxybenzoate Propyl Hydroxybenzoate acid.
of the British Pharmacopoeia. Prepare immediately before use.
Propy1ene Glycol Of the British Pharmacopoeia .
Pyridine-3-carboxaldehyde Nicotinaldehyde; Quercitrin Quercetin 3-L-rhamnopyranoside; 3-[(6-deoxy-

C 6H sNO = 107.1 (500-22-1) cH-mannopyranosyl) oxy] -2- (3 ,4-dihydroxyphenyl)-5, 7-
General reagent grade of commerce. dihydroxy-4H-l-benzopyran-4-one; quercitroside;
Weight per mL, 1.14 g. C21H20011 = 448 .4 (522-12-3)
Yellow crystals, practically insoluble in cold water, soluble in
Pyridinium Hydrobromide Perbromide Pyridinium
ethanol (96%).
tribromide(I-); CSH6Br3N = 319.8 (39416-48-3)
Meltingpoint, 176 0 to 179".
Red crystals.
Pyrid-2-ylamine See 2-Pyridylamine.
Chromatography Thin-Iayer chromatography (2.2.27) as
prescribed in the monograph Goldenrod (1892): apply 20 ~lL
2-Pyridylamine 2-Aminopyridine; pyrid-2-ylamine; pyrid- of the solution; after spraying, the chromatogram shows a
2-amine C SH 6N 2 = 94.1 (504-29-0) yellowish-brown fluorescent zone with an RF of about 0.6.
General reagent grade of commerce. Store at a temperature of 2° to 8°.
Melting point, about 58°; boiling point, about 210°. Quillaia Saponins, Purified
Pyridylazonaphthol PAN; 1-(2-pyridylazo )-2-naphthol; A mixture of related saponins obtained from the bark of
C 1sH ll N 30 = 249.3 (85-85-8) Quillaja saponaria Molina s.l.
General reagent grade of commerce. Chromatography Thin-Iayer chromatography (2.2.27) as
A brick red powder; melting point, about 138°. prescribed in the monograph Quillaia bark (1843): apply
Pyridylazonaphthol Solution A 0.1 % w/v solution in 5 ¡.tL of the solution; after treating with a 10% v/v solution
absolute ethanol. of sulfuric acid in methanol, heat at 120° for 5 minutes and
Complies with the following test. examine in daylight; the chromatogram shows 3 principal
zones in the upper part of the middle third.
Sensitivity to copper To 50 mL of water add 10 mL of
acetate buffer pH 4.4, 0.1 mL of 0.02M disodium edetate VS Quinaldine Red 2- [2- [4-(Dimethylamino)phenyl] ethenyl]-
and 0.25 mL of the reagent being examined; a yellow colour l-ethylquinolinium iodide,
is produced. Add 0.15 mL of a 0.5% w/v solution of 2-( 4-dimethylaminostyryl)quinoline ethiodide;
copper(n) sulfate; the colour changes to vio let. C21H23IN2 = 430.3 (117-92-0)
4-(2-Pyridylazo)resorcinol Monosodium Salt Dark bluish-black powder, sparingly soluble in water, freely
CllHsN3Na02,H20 = 255.2 (16593~81-0) soluble in ethanol (96%).
General reagent grade of commerce. Quinaldine Red Solution Dissolve 0.1 g of quinaldine red
in methanol R and dilute to 100 mL with the same solvento
Pyrocatechol See Catecho!'
Colour change pH 1.4 (colourless) to pH 3.2 (red).
Pyrogallol Benzene-l,2,3-triol; C 6H 60 3 = 126.1
Quinhydrone C 12H IO 0 4 = 218 .2 (106-34-3)
Equimolecular compound of 1,4-benzoquinone and
White crystals becoming brownish on exposure to air and
hydroquinone.
light; melting point, about 131 0.
Dark green, lustrous crystals or a crystalline powder, slightly
soluble in water, sparingly soluble in hot water, soluble in
Pyrogallol Solution, A1kaline Dissolve 0.5 g of pyrogallol ethanol (96%) and in concentrated ammonia.
in 2 mL of carbon dioxide-free water; dissolve 12 g of
potassium hydroxide in 8 mL of carbon dioxide-free water.
Mix the two solutions immediately before use. Quinidine (S)-(6-Methoxyquinol-4-yl) [(2R,4S,5R)-5-
vinylquinuclidin-2-yl]methanol;
Pyrrolidine C 4 H 9 N = 71.1 (123-75-1)
C2oH24N202 = 324.4 (56-54-2)
Content, minimum 99 % of C 4 H 9 N; boiling point, 8r to
White or almost white crystals, very slightly soluble in water,
88°.
sparingly soluble in ethanol (96%), slightly soluble in
2-Pyrrolidone Pyrrolidin-2-one; methano!.
C 4 H 7NO = 85 .1 (616-45-5)
[a]~o, about + 260, determined on a lO giL solution in
General reagent grade of commerce. anhydrous ethanol; melting point, about 172°.
Content, minimum 98.0%; liquid aboye 25°; d~5, 1.116 . Store protected from light.
Pyruvic Acid 2-0xopropanoic acid; Quinidine Sulfate Quinidine sulphate;
C 3 H 4 0 3 = 88. 1 (127-17-3) C4oH4SN404,HzS04,2H20 = 783.0 (6591-63-5)
General reagent grade of commerce. Of the British Pharmacopoeia.
d~g, about 1.267; n~, about 1.413; boiling point, about Quinine (R)-( 6-Methoxyquinol-4-yl) [(2S,4S,5R)-5-
165°. vinylquinuclidin-2-yl]methanol;
Quercetin Dihydrate 2-(3,4-Dihydroxyphenyl)-3,5,7- C2oH2~202 = 324.4 (130-95-0)
trihydroxy-4H-l-benzopyran-4-one; White or almost white, microcrystalline powder, very slightly
ClsHIO07,2H20 = 338.2 soluble in water, slightly soluble in boiling water, very soluble
Yellow crystals or yellowish powder, practically insoluble in in anhydrous ethano!.
water, soluble in acetone and in methano!.
[al~, about -167, determined on a 10 gIL solution in
Water (2.5.12) Maximurn 12.0%, determined on 0.100 g. anhydrous ethanol; melting point, 175°.
monograph Ginkgo leaf (1828).
Quinine Hydroch1oride
Content, minimum 90% (anhydrous substance) calculated by
C2oH2~z02,HCI,2H20 = 396.9 (6119-47-7)
the normalisation procedure.
Store protected from light. Of the British Pharmacopoeia.
V-AllO Appendix 1 A 2014
Quinine Sulfate Quinine sulphate; L-Rhamnose C6H120S,H20 = 182.2 (6155-35-7)

C4oH4SN404,H2S04,2H20 = 783.0 (6119-70-6) General reagent grade of commerce.
Of the British Phannacopoeia. A white, crystalline powder; melting point, about 96°; [a]~,
Quinoline C 9 H 7N = 129.2 (91-22-5) + 7.8 to +8.3 (5% w/v in water containing about 0.05 % of
General reagent grade of commerce, suitable for phosphate NH3 ) ·
assay. Rhaponticin Rhapontin; 4'-methoxystilbene-3,3',5-triol
A pale yellow liquid; n~, 1.624 to 1.627. 3-glucoside; CZ1Hz409 = 420.4 (155-58-8)
Complies with the following requirement. General reagent grade of commerce.
Tarry impurities Dissolve 1.0 g in a mixture of 2.5 mL Yellowish grey crystalline powder.
of water and 2.5 mL of hydroehlone acid. Add 3 mL of water Complies with the following test.
and 1 mL of potassium ehromate solution and heat to boiling. Homogeneity Carry out the test for Rheum rhapontieum
The solution shows no darkening and no black deposit. described in the monograph for Rhubarb. The
Store protected from light. chromatogram obtained with the reference solution shows
Quinoline Solution Dissolve 50 mL of quinoline in a only one principal spot.
mixture of 60 mL of hydroehlone acid and 300 mL of water Rhodamine 6 G 9-[2-(Ethoxycarbonyl)phenyl]-3,6-
previously heated to 70°, cool and filter. bis( ethylamino )-2, 7-dimethylxanthenylium chloride;
Rabbit Erythrocyte Suspension Prepare a 1.6% w/v C2sH31ClNz03 = 479.0 (989-38-8)
suspension of rabbit erythrocytes in the following manner. Colour Index No. 45160.
Defibrinate 15 mL of freshly drawn rabbit blood by shaking General reagent grade of commerce.
with glass beads, centrifuge at 2000 g for 10 minutes and
Brownish-red powder.
wash the erythrocytes with three 30-rnL quantities of a
0.9% w /v solution of sodium chloride. Dilute 1.6 mL of the
Rhodamine B CI 45170; basic violet 10;
suspension of erythrocytes to 100 mL with a mixture of C2sH31ClNz03 = 479.0 (81-88-9)
1 volume of phosphate buffer pH 7.2 and 9 volumes of a General reagent grade of commerce.
0 .9% w/v solution of sodium ehlonde. Green crystals or a reddish violet powder.
Rabies Antiserum, Fluorescein-con;ugated Ribose D-Ribose; C SH lO O S = 150.1 (50-69-1)
Immunoglobulin fraction with a high rabies antibody titre, General reagent grade of commerce.
prepared from the sera of suitable animals that have been
immunised with inactivated rabies virus. The
immunoglobulin is conjugated with ftuorescein Ricinoleic Acid 12-Hydroxyoleic acid;
isothiocyanate. Cl sH 3403 = 298.5 (141-22-0)
Raclopride Tartrate Raclopride L-tartrate; General reagent grade of commerce.
Cl9Hz6ClzN209 = 497.3 (98185-20-7) d~g, about 0.942; n~o, about 1.472; melting point, about
A white solid, sensitive to light, soluble in water. 285 °, with decomposition.
[a]~z : + 0.3, detennined on a 3 gIL solution. Melting point, Rosmarinic Acid ClsH160S = 360.3 (20283-92-5)
about 141 °. Melting point, 170° to 174°.
Rapeseed Dil Of the British Pharmacopoeia. Ruscogenins Mixture of neoruscogenin
Reducing Mixture Hydrazine reducing mixture (C 27 H 40 0 4 = 428.6) and ruscogenin (Cz7H4204 = 430.6)
Grind 20 mg of potassium bromide, 0.5 g of hydrazine sulfate General reagent grade of commerce.
and 5 g of sodium ehlonde in the order listed to obtain a Ruseogenins used in liquid chromatography complies with the
homogeneous mixture. following test.
Reichstein's Substance S C 21H3004 = 346.5 (152-58-9) Assay Carry out the method described in the monograph
Content, minimum 95.0%; melting point, about 208°. for Butcher's Broom. The content is not less than 90% of
ruscogenins of which at least 60% consists of neoruscogenin,
Reserpine Methyl 11, 1711-dimethoxy-18~- [(3,4,5-
calculated by the normalisation procedure.
trimethoxybenzoyl)oxy]-3 ~,2011-yohimban-16~-carboxylate;
C33H4oN20 9 = 609 (50-55-5) The reagent from Vinyals laboratory has been found suitable.
General reagent grade of cornrnerce. Ruthenium Red Arnrnoniated ruthenium oxychloride;
[(NH3)sRuORu(NH3)40Ru(NH3)S]CI6,4H20 = 858
Resin for Reversed-phase Ion Chromatography
(11103-72-3)
A neutral, macroporous, high specific surface are a with a
Microscopical staining grade of commerce.
non-polar character resin consisting of polymer lattice of
polystyrene cross-linked with divinylbenzene. A brownish red powder.
Resorcinol Benzene-1,3-diol; C 6H 60 2 = 110.1 (108-46-3) Ruthenium Red Solution A 0.08% w /v solution of
ruthenium red in lead aeetate solution.
Rutin Vitamin P; rutoside; 3-(O-6-DeoXY-I1-L-
Colourless crystals or crystalline powder; melting point,
mannopyranosyl-(1->6)-~-D-glucopyranosyloxy)-2-(3,4-
about 111 °.
dihydroxyphenyl)-5,7 -dihydroxy-4H-chromen-4-one;
Resorcinol Reagent To 80 mL of hydroehlone acid add 3,3 ' ,4' ,5, 7 -pentahydroxyftavone 3-rutinoside;
10 rnL of a 2% w/v solution of resoreinol and 0.25 mL of a C27H30016,3H20 = 665 (153-18-4)
2.5 % w/v solution of copper(lI) sulfate and dilute to 100 mL
with water. Prepare the solution at least 4 hours before use.
A yellow, crystalline powder, darkening in light, very slightly
Store at 2° to 8° and use within 1 week.
soluble in water, soluble in about 400 parts of boiling water,
Rharnnose See L-Rhamnose.
slightly soluble in alcohol, soluble in solutions of the alkali Povidone (0685); the chromatogram shows only one principal
hydroxides and in arnmonia. spot.
Melting point, about 210°, with decomposition; a solution in Salicylic Acid C 7H 60 3 = 138. 1 (69- 72-7)
ethanol (96%) exhibits absorption maxima at 259 nm and Of the British Pharmacopoeia .
362 nm, Appendix II B. Saline Solution A 0.9 % w/v solution of sodium ehlo/'ide in
Store protected from light. freshly prepared water, sterilísed by heating in an autoclave.
Sabinene Thuj-4(l O)-ene; 4-Methylene-1- Salvianolic Acid B (2R)-2-[[(2E) -3-[(2S,3S)-3-[[(lR)- I-
isopropylbicyclo[3.1.0]hexane; C lO HI 6 = 136.2 (3387-41-5) Carboxy-2-(3 ,4-dihydroxyphenyl) ethoxy] carbonyl]-2- (3,4-
A colourless, oily liquido dihydroxyphenyl)-7-hydroxy-2,3-dihydrobenzofuran-4-
Sabinene used in gas ehromatography eomplies with the following yl] prop-2-enoyl] oxy]-3-(3,4-dihydroxyphenyl)propanoic acid;
additional test. C36H 3001 6 = 719 (12152 1-90-2)
Assay Gas chromatography (2.2.28) as prescribed in the Sand White or slightly greyish grains of silica with a
monograph Bitter-orange-flower oil (1175). particle size between 150 ¡.lm and 300 pm.
Test solution The substance to be examined. Santonin ClsHIS03 = 246.3 (48 1-06-1)
Content, minimum 95 .0%, calculated by the normalisation General reagent grade of commerce.
procedure. M elting point, 174° 10 176°; [a]g, 173 in ethano!.
Saccharin C 7H sN0 3S = 183.2 (81-07-2) Chromatography Carry out Identification test C in the
General reagent grade of commerce. monograph for Arnica Flower using a 0.025% w/v solution
of the reagent being examined in methanol. The
chromatogram obtained with 1O ~IL of the solution shows a
Sacchar~ Sodium C7H4NNa0 3S = 205.2 (128-44-9)
quenching zone with an Rf value of abo ut 0.5. Spray the
Of the British Pharmacopoeia. plate with anisaldehyde solution and examine while heating at
Safrole 4-Allyl-1,2-(methylenedioxy) benzene, 5-(Prop-2- 105 e for 5 10 10 minutes. In daylíght the quenching zone is
enyl)-1,3-benzodioxole; CIOHIOOz = 162.2 (94-59-7) at first a yellow zone that quickly changes 10 a vio1et-red
Colourless or slightly yellow, oily liquid, with the odour of zone.
sassafras, insoluble in water, very soluble in ethanol (96%) , Saraftoxacin Hydrochloride 6-Fluoro-l-
miscible with hexane. (4-fiuorophenyl)-4-oxo-7 -piperazin-l-yl-l ,4-dihydroquinoline-
d~g, 1.09510 1.096; n~, 1.537 to 1.538; boiling point, 232
0 3-carboxylic acid hydrochloride; CZOHI SCIFzN303 = 421.8
0
to 234 ; freezing point, about 11 0 . (91296-87-6)
Safrole used in gas ehromatography complies with the follo wing General reagent grade of commerce.
additional test. Schisandrin Schisandrol A. Wuweizichun A;
Assay Gas chromatography (2.2.28) as prescribed in the (6S, 7S, 12aRa)-5,6, 7,8-T etrahydro-1 ,2,3, 1O, 11, 12-
monograph Cinnamon bark oil, Ceylon (1501). hexamethoxy-6, 7 -dimethyldibenzo [a, e] cyclooctan-6-01;
C 24 H 32 0 7 = 432.5 (7432-28-2)
procedure. White or almost white, crystalline powder.
Salicin 2-(Hydroxymethyl)phenyl-~-D Schisandrin used in the assay in the monograph Sehisandra
glucopyranoside;salicoside; C 13 H 1S0 7 = 286.3 (138-52-3) fruit (2428) complíes with the following additional test.
[a]~, - 62.5 ± 2; melting point, 199° to 201 °. Assay Liquid chromatography (2.2.29) as prescribed in the
monograph Sehisandra fruit (2428).
monograph Willow bark (1583) at the concentration of the Content, minimum 95 %, calculated by the normalisation
reference solution. procedure.
Content, minimum 99.0%, calculated by the normalisation Store in an airtight container, at - 20 0 or below.
procedure. y-Schisandrin Schisandrin B. Wuweizisu B; rae-
Salicylaldehyde 2-H ydroxybenzaldehyde; (6R, 7S, 13aRa)-1,2,3, 13-Tetramethoxy-6, 7-dimethyl-5,6, 7 ,8-
C7H60 Z = 122.1 (90-02-8) tetrahydrobenzo[3,4] cycloocta [1 ,2-j] [1 ,3]benzodioxole;
CZ3H 2S0 6 = 400 .5 (61281-37-6)
Clear, colourless, oily liquido
White or almost white, crystalline powder.
d~g, about l.l67; n6°, about 1.574; boiling point, about
196°; m elting point, about -r. Store in an airtight container, at - 20° or below.
Salicylaldehyde Azine 2,2 '-Azinodimethyldiphenol; Sc1areol (IR,2R,4aS,8aS)-I-[(3R)-3-Hydroxy-3-
CI4HI2NzOz = 240 .3 (959-36-4) methylpent-4-enyl]-2,5,5,8a-
tetramethyldecahydronaphthalen-2-ol;
D issolve 0.30 g of hydrazine sulfate in 5 mL of water, add
C ZOH 360 = 308.5 (5 15-03-7)
1 mL of glacial aeetie acid and 2 mL of a freshly prepared
20% v/v solution of salicylaldehyde in 2-pl'Opanol. Mix, allow Odourless crystals.
10 stand until a yellow precipate is formed. Shake with two [a]~, 6.7, determined with a solution in anhydrous ethanol;
quantities, each of 15 mL, of methylene ehloride. Combine the boiling pointl9mm, 218° to 220°; melting point, 96° to 98' .
organic layers and dry over anhydrous sodium sulfate. D ecant Sclareol used in the ehromatographie profile test in the monograph
or filter the solution and evaporate 10 dryness . Recrystallise Clary sage oil (1850) eomplies with the following additional test.
from a mixture of 40 volumes of methanol and 60 volumes of Assay Gas chromatography (2.2.28) as prescribed in the
toluene with cooling. Dry the crystals in vacuo. m onograph Clary sage oil (1850).
Content, minimum 97%, calculated by the normalisation
Chromatography Thin-layer chromatography (2.2.27) as procedure.
prescribed in the test for hydrazine in the monograph
V-Al12 Appendix 1 A 2014
Scopoletin 7-Hydroxy-6-methoxy-2H-1-benzopyran-2-one; White or yellowish powder, practically insoluble in water,

7-Hydroxy-6-methoxycoumarin; C lO H s 0 4 = 192.2 (92-61-5) soluble in acetone and in methanol.
Faintly beige, fine crystals. Silibinin used in the assay of Milk-thistle fruit (1860) complies
Melting point, 202° to 208°. with the following additional test.
SDS-PAGE Running Buffer Dissolve 151.4 g of Assay Liquid chromatography (2.2.29) as prescribed in the
tris(hydroxyrnethyl)aminomethane, 721.0 g of glycine and monograph Milk-thistle fruit (1860).
50.0 g of sodium lauryl sulfate in water and dilute to 5000 mL Test solution Dissolve 5.0 mg of silibinin, dried in vacuo,
with the same solvent. Irnmediately before use, dilute to in methanol and dilute to 50.0 mL with the same solvent.
10 times its volume with water and mix. Measure the pH Silibinin A and silibinin B content, minimum 95.0%,
(2.2.3) of the diluted solution. The pH is between 8.1 and calculated by the normalisation procedure.
8.8. Silica Gel A fine, white, homogeneous powder of an
SDS-PAGE Sample Buffer (Concentrated) Dissolve average partic1e size of about 15 ~ containing a suitable
1.89 g of tris(hydroxymethyl)aminomethane, 5.0 g of sodium binding agent.
lauryl sulfate and 50 mg of bromophenol blue in water.
Silica Gel n-Acceptorht-Donor for Chiral Separations
Add 25.0 mL of glycerol and dilute to 100 mL with water.
A very finely divided silica gel for chromatography consisting
Adjust the pH to 6.8 with hydrochloric acid, and dilute to
of spherical partic1es to which 1-(3,5-dinitrobenzamido)-
125 mL with water.
1,2,3,4-tetrahydrophenantrene has been covalently bound,
SDS-PAGE Sample Buffer for Reducing Conditions showing both n-electron acceptor and n-electron donor
(Concenttated) Dissolve 3.78 g of characteristics. The partic1e size and the configuration are
tris(hydroxymethyl)aminomethane, 10.0 g of sodium dodecyl
indicated after the name of the reagent in the tests where it is
sulfate and 100 mg of bromophenol blue in water.
used.
Add 50.0mL of glycerol and dilute to 200 mL with water.
Silica Gel AD for Chiral Separation
Add 25.0 mL of 2-mercaptoethanol. Adjust to pH 6.8 with
hydrochloric acid, and dilute to 250.0 mL with water. A very finely divided silica gel for chromatography (5 }lm)
Altematively, dithiothreitol may be used as reducing agent coated with the following derivative:
instead of 2-mercaptoethanol. In this case prepare the sample
ro c",~R
y
H
buffer as follows: dissolve 3.78 g of H3C N, c /
R
tris(hydroxymethyl)aminomethane, 10.0 g of sodium dodecyl I 11
t
sulfate and 100 mg of bromophenol blue in water. Add 50.0 mL ~ o
OR
of glycerol and dilute to 200 mL with water. Adjust to pH 6.8 CH 3
OR OR
with hy drochloric acid, and dilute to 250.0 mL with water.
Immediately before use, add dithiothreitol to a final
concentration of 100 mM. Silica Gel AGP for Chiral Chromatography
Selenious Acid Selenous acid; selenic(Iv) acid; A very finely divided silica gel for chromatography consisting
H2Se03 = 129.0 (7783-00-8) of spherical partic1es coated with a1- acid glucoprotein. The
Deliquescent crystals, freely soluble in water. partic1e size is indicated after the name of the reagent in the
tests where it is used.
Silica Gel, Anhydrous (112926-00-8)
Selenium Se = 79.0 (7782-49-2)
Partly dehydrated polymerised, amorphous silicic acid,
Brown-red or black powder or granules, practically insoluble
absorbing at 20° about 30% of its mas s of water. Practically
in water and in ethanol (96%), soluble in nitric acid.
insoluble in water, partly soluble in solutions of sodium
Melting point, about 220°. hydroxide. It contains a suitable indicator for detection of the
Selenium Dioxide Se02 = 111.0 (7446-08-4) humidity status, for which the colour change from the
General reagent grade of commerce. hydrated to anhydrous form is given on the label.
Semicarbazide Acetate Solution Triturate 2.5 g of Silica Gel BC for Chiral Chromatography
semicarbazide hydrochloride with 3.3 g of sodium acetate, add A very finely divided silica gel for chromatography (5 }lm)
10 mL of methanol, mix, transfer to a fiask with the aid of coated with ~-cyc1odextrin. Higher selectivity may be
20 mL of methanol and allow to stand at a temperature of obtained when cyc10dextrin has been derivatized with
about 4° for 30 minutes; filter and add sufficient methanol to propylene oxide.
produce 100 mL. Silica Gel for Chiral Chromatography, Urea Type A
Semicarbazide Hydrochloride very finely divided silica gel (5 }lm) coated with the following
CHsN 30,HCI = 111.5 (563-41-7) derivative:
A white, crystalline powder; melting point, about 175°, with
decomposition.
Serine C 3H 7N0 3 = 105 .1 (56-45-1)
L-Serine See serine. Silica Gel for Chromatography
Sialic Acid See N-Acetylneuraminic acid. A very finely divided (3 }lm-10 }lm) silica gel. The particle
Silibinin Silybin; (2R,3R)- 3,5, 7-Trihydroxy-2-[ (2R,3R)-3- size is indicated after the name of the reagent in the tests
(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3- where it is used.
dihydro-1,4-benzodioxin-6-yl]-2,3-dihydro-4H-1-benzopyran- Fine, white or almost white, homogeneous powder,
4-one; C2sH2201 0 = 482.4 (22888-70-6) practically insoluble in water and in ethanol (96%).
2014 Appendix 1 A V-Al13
Silica Gel for Chromatography, AIkyl-bonded for use any interaction with basic compounds, it is carefully end-
with Highly Aqueous Mobile Phases capped 10 cover most of the remaining silanol groups.
A very finely divided silica gel with bonded alkyl groups The particle size is indicated after the name of the reagent in
suitable for use with highly aqueous mobile phases. the tests where it is used.
Silica Gel for Chromatography, AIkyl-bonded for use Fine, white or almost white, homogenous powder, practically
with Highly Aqueous Mobile Phases, End-capped insoluble in water and in ethanol (96%).
A very finely divided silica gel with bonded alkyl groups Silica Gel for Chromatography, Crown-ether
suitable for use with highly aqueous mobile phases. To Stationary phase for liquid chromatography.
minimise any interaction with basic compounds it is carefully Crown ether coated on silica gel.
end-capped to cover most of the remaining silanol groups. Silica Gel for Chromatography, Cyanosilyl
The particle size is indicated after the name of the reagent in A very finely divided silica gel chemically modified at the
the tests where it is used. surface by the bonding of cyanosilyl groups. The particle size
Silica Gel for chromatography, Amidohexadecylsilyl is indicated after the name of the reagent in the tests where it
A very finely divided silica gel with a fine particle size, is used.
chemically modified at the surface by the bonding of Fine, white or almost white, homogeneous powder,
amidohexadecylsilyl groups. The particle size is indicated practically insoluble in water and in ethanol (96%).
after the name of the reagent in the test where it is used. Silica Gel for Chromatography, Di-
Silica Gel for Chromatography, Aminohexadecylsilyl isobutyloctadecylsilyl
A very finely divided (3-10 !lm) silica gel with a fine particle A very finely divided silica gel chemically modified at the
size chemically modified at the surface by the bonding of surface by the bonding of di-isobutyloctadecylsilyl groups.
aminohexadecylsilyl groups. The particle size is indicated The particle size is indicated after the name of the reagent in
after the name of the reagent in the test where it is used. the tests where it is used.
Fine, white or almost wrute, homogeneous powder, Silica Gel for Chromatography,
practically insoluble in water and in ethanol (96%). Düsopropylcyanopropylsilyl
Silica Gel for Chromatography, A very finely divided silica gel chemically modified at the
Aminopropylmethylsilyl surface by the bonding of diisopropylcyanopropylsilyl groups.
Silica gel with a fine particle size (between 3 ¡.tm and ~O ¡.tm), The particle size is indicated after the name of the reagent in
chemically modified by bonding aminopropylmethylsilyl which the test is used.
groups on the surface. The particle size is indicated after the Silica Gel for Chromatography, Dimethyloctadecylsilyl
name of the reagent in the tests where it is used. A very finely divided silica gel (3 !lm-lO !lm), chemically
Fine, white or almost wrute, homogeneous powder, modifiedat the surface by the bonding of
practically insoluble in water and in ethanol (96%). dimethyloctadecylsilyl groups.The particle size is indicated
Silica Gel for Chromatography, Aminopropylsilyl after the name of the reagent inthe tests where it is used.
Silica gel with a fine particle size (between 3 !lm and 10¡.tm), Fine, white or almost white, homogeneous powder,
chemically modified by bonding aminopropylsilyl groups on practically insoluble in water and in ethanol (96%). Irregular
the surface. The particle size is indicated after the name of particle size.
the reagent in the tests where it is used. Specific surface area, 300 m2g- 1.
Fine, wrute or almost white, homogeneous powder, Silica Gel for Chromatography, Diol
practically insoluble in water and in ethanol (96%). Spherical silica particles 10 which dihydroxypropyl groups are
Silica Gel for Chromatography Rl, Aminopropylsilyl bonded. Pore size 10 nm.
Silica gel with a particle size of about 55 ¡.tm, chemically Silica Gel for Chromatography, Dodecylsilyl, End-
modified by bonding aminopropylsilyl groups on the surface. capped
Silica Gel for Chromatography, Amylose-derivative of A very finely divided silica gel, chemically modified at the
A very finely divided (10 !lm) silica gel, chemically surface by the introduction of dodecylsilyl groups.
modifiedat the surface by the bonding of an amylose To minimise any interaction with basic compounds, it is
derivative. Theparticle size is indicated after the name of the carefully end-capped to cover most of the remaining silanol
reagent in thetest where it is used. groups.
Fine, wrute or almost white, homogenous powder, practically Silica Gel for Chromatography, Hexadecylamidylsilyl
insoluble in water and in ethanol (96%). A very finely divided (5 !lm) silica gel, chemically modified at
Silica Gel for Chromatography, Butylsilyl the surface by the introduction of
A very finely divided silica gel (3 ¡.1m-lO !lm), chemically hexadecylcarboxamidopropyldimethylsilyl groups.
modified at the surface by the bonding of butylsilyl groups. Silica Gel for Chromatography, Hexadecylamidylsilyl,
The particle size is indicated after the name of the reagent in End-capped
the tests where it is used. A very finely divided (5 !lm) silica gel, chemically modified at
Fine, white or almost white, homogeneous powder, the surface by the introduction of
practically insoluble in water and in ethanol (96%). hexadecylcarboxamidopropyldimethylsilyl groups. To
Spheroidal silica, 30 nm; pore volume, 0.6 cm 3g- 1; specific minimise any interaction with basic compounds it is carefully
surface area, 80 m2g- 1. end-capped 10 cover most of the remaining silanol groups.
Silica Gel for Chromatography, Butylsilyl, End-capped Silica Gel for Chromatography, Hexylsilyl
A very finely divided silica (3-10 ¡.1m), chemically modified at A very finely divided (3 !lm-lO !lm) silica gel, chemically
the surface by the bonding of butylsilyl groups. To minimise modified at the surface by the bonding of hexylsilyl groups.
The particle size is indicated after the name of the reagent in Fine, white or almost white, homogeneous powder,
the tests where it is used. practically insoluble in water and in ethanol (96%) .
Fine, white or almost white, homogeneous powder, Silica Gel for Chromatography, Octadecylsilyl
practically insoluble in water and in ethanol (96%). A very finely divided (3 ).1m-lO ).1m) silica gel, chemically
Silica Gel for Chromatography, Hexylsilyl, End-capped modified at the surface by the bonding of octadecylsilyl
A very finely divided (3-10 ).1m) silica gel, chemically groups. The particle size is indicated after the name of the
modified at the surface by the bonding ofhexylsilyl groups. reagent in the tests where it is used.
To minimise any interaction with basic compounds it is Fine, white or almost white, homogeneous powder,
carefully end-capped to cover most of the remaining silanol practically insoluble in water and in ethanol (96%).
groups. The particle size is indicated after the name of the Silica Gel for Chromatography, Octadecylsilyl, Base-
reagent in the tests where it is used. deactivated
A fine, white or almost white, homogeneous powder, A very finely divided (3 ).1m-lO 11m) silica gel, pretreated
practically insoluble in water and in ethanol (96%). before the bonding of octadecylsilyl groups by careful
Silica Gel for Chromatography, Human Albumin washing and hydrolysing most of the superficial siloxane
Coated bridges to minimise the interaction with basic components.
A very finely divided (3 ).1m to 10 ).1m) silica gel, chemically The particle size is indicated after the name of the reagent in
modified at the surface by the bonding of human albumin. the tests where it is used.
The particle size is indicated after the name of the reagent in Fine, white or almost white, homogeneous powder,
the tests where it is used. practically insoluble in water and in ethanol (96%).
White or almost white, fine, homogeneous powder. Silica Gel for Chromatography, Octadecylsilyl, End-
Silica Gel for Chromatography, Hydrophllic capped
A very finely divided (3 ).1m-lO ).1m) silica gel whose surface A very finely divided (3 I1m-1 O ~lm) silica gel, chemically
has been modified to provide hydrophilic characteristics. modified at the surface by the bonding of octadecylsilyl
The particle size may be stated after the name of the reagent groups. To minimise any interaction with basic compounds it
in the tests where it is used. is carefully end-capped to cover most of the remaining silanol
Silica Gel for Chromatography, Methylsilyl groups. The particle size is indicated after the name of the
reagent in the tests where it is used.
Liquid chromatographic reagent grade of commerce.
Fine, white or almost white, homogenous powder, practically
A very finely divided silica gel (3 to 10 ).1m) chemically insoluble in water and in ethanol (96%).
modified at the surface by the introduction of methylsilyl
Silica Gel for Chromatography, Octadecylsilyl, End-
groups. The particle size is specified after the name of the
capped, Base-deactivated
reagent in tests where it is used.
A very finely divided (3 11m-lO 11m) silica gel with a pore size
Silica Gel for Chromatography, Nitrile of 10 nm and a carbon loading of 16%, pre-treated before
A very finely divided silica gel, chemically modified at the the bonding of octadecylsilyl groups by washing and
surface by the bonding of cyanopropylsilyl groups. hydrolysing most of the superficial siloxane bridges.
The particle size is indicated after the name of the reagent in To further minimise any interaction with basic compounds it
the test where it is used. is carefully end-capped ro cover most of the remaining silanol
Fine white or almost white, homogenous powder, practically groups. The partic1e size is indicated after the name of the
insoluble in water and in ethanol (96%). reagent in the test where it is used.
Silica Gel for Chromatography, Nitrile, End-capped Fine, white or almost white, homogeneous powder,
A very finely divided silica gel, chemically modified at the practically insoluble in water and in ethanol (96%).
surface by the bonding of cyanopropylsilyl groups . To Silica Gel for Chromatography, Octadecylsilyl,
minimise any interaction with basic components it is carefully Monolithic
end-capped ro cover most of the remaining silanol groups. Monolithic rods of highly porous (greater than 80%) metal-
The particle size is indicated after the name of the reagent in free silica with a bimodal pore structure, modified at the
the test where it is used. surface by the bonding of octadecylsilyl groups.
A fine, white or almost white, homogenous powder, Silica Gel for Chromatography, Octadecylsilyl, with
practically insoluble in water and in anhydrous ethano!. Embedded Polar Groups, End-capped
Silica Gel for Chromatography, A very finely divided silica gel (3-10 11m) . The partic1es are
4-Nitrophenylcarbamidesilyl based on a mixture of silica chemically modified at the
surface by the bonding of octadecylsilyl groups and silica
A very finely divided silica gel, chemically modified at the
chemically modified with a reagent providing a surface with
surface by bonding of 4-nitropheny1carbamide groups. The
chains having embedded polar groups. Furthermore, the
particle size. is indicated after the na me of the reagent in the
packing material is end-capped. The partic1e size is indicated
tests where it is used.
after the name of the reagent in the tests where it is used.
Fine, white or almost white, homogeneous powder, Silica Gel for Chromatography, Octadecylsilyl, with
practically insoluble in water and in ethanol (96%).
Polar Incorporated Groups, End-capped
Silica Gel for Chromatography, A very finely divided silica gel (3-10 11m). The partic1es are
Octadecanoylaminopropylsilyl based on silica, chemically modified with a reagent providing
A very finely divided (3 ).1m-lO ).1m) silica gel, chemically a surface with chains having polar incorporated groups and
modified at the surface by the bonding of aminopropylsilyl terminating octadecyl groups. Furthermore, the packing
groups which are acylated with octadecanoyl groups. material is end-capped. The partic1e size is indicated after the
The particle size is indicated after the name of the reagent in name of the reagent in the tests where it is used.
the tests where it is used. A fine, white or almost white, homogeneous powder.
2014 Appendix 1 A V-A1l5
Silica Gel for Chromatography, Octylsilyl A very finely divided silica gel, chemically modified at the
A very finely divided (3 )lm-1 O ~lm) silica gel, chemically surface by the bonding of phenyl groups. The partic1e size is
modified at the surface by the bonding of octylsilyl groups. indicated after the name of the reagent in the tests where it is
The partic1e size is indicated after the name of the reagent in used.
the tests where it is used . Silica Gel for Chromatography, Phenylethyl, End-
Fine, white or almost white, homogeneous powder, capped
practically insoluble in water and in ethanol (96%) . Liquid chromarographic reagent grade of commerce.
Silica Gel for Chromatography, Octylsilyl, Base- A very finely divided silica gel (2.5 ro 10 ¡lm) chemically
deactivated modified at the surface by the introduction of ether-linked
A very finely divided (3 ¡lm-10 )lm) silica gel, pretreated phenyl groups. To minimise any interaction with basic
before the bonding of octylsilyl groups by careful washing compounds ir is carefully end-capped to cover most of the
and hydrolysing most of the superficial siloxane bridges to remaining silanol groups. The particle size is specified after
minimise the interaction with basic components. The partic1e the name of the reagent in tests where it is used.
size is indicated after the name of the reagent in the tests Silica Gel for Chromatography, PhenyIhexylsilyl
where ir is used. A very finely divided silica gel, chemically modified at the
Fine, white or almost white, homogeneous powder, surface by the bonding of phenylhexyl groups. The particle
practically insoluble in water and in ethanol (96%). size is indicated after the name of the reagent in the tests
Silica Gel for Chromatography, Octylsilyl, End-capped where it is used.
A very finely divided (3 ¡lm-10 ¡lm) silica gel, chemically Silica Gel for Chromatography, PhenyIhexylsilyl, End-
modified at the surface by the bonding of octylsilyl groups . capped
To minimise any interaction with basic compounds, it is A very finely divided silica gel (3 ¡lm), chemically modified at
carefully end-capped ro cover most of the remaining silanol the surface by the bonding of phenylhexylsilyl groups.
groups. The partic1e size is indicated after the name of the To minimise any interaction with basic compounds, it is
reagent in the tests where it is used. carefully end-capped to cover most of the remaining silanol
Fine, white or almost white, homogeneous powder, groups. The partic1e size is indicated after the name of the
practically insoluble in water and in ethanol (96%). reagent in the tests where it is used.
Silica Gel for Chromatography, Octylsilyl, End- Silica Gel for Chromatography, Phenylsilyl
capped, Base-deactivated A very finely divided silica gel, chemically modified at the
A very finely divided (3 ¡lm-10 ¡lm) silica gel, pre-treated surface by the bonding of phenyl groups. The particle size is
before the bonding of octylsilyl groups by washing and indicated after the name of the reagent in the tests where it is
hydrolysing most of the superficial siloxane bridges. used.
To further minimise any interaction with basic compounds it Silica Gel for Chromatography, Phenylsily1, End-
is carefully end-capped ro cover most of the remaining silanol capped
groups. The particle size is indicated after the name of the A very finely divided (5 to 10 ¡lm) silica gel, chemically
reagent in the test where it is used. modified at the surface by the bounding of phenyl groups.
Fine, white or almost white, homogeneous powder, To minimise any interaction with basic compounds it is
practically insoluble in water and in ethanol (96%). carefully end-capped to cover most of the remaining silanol
Silica Gel for Chromatography, Octylsilyl, with Polar groups. The particle siú is indicated after the name of the
Incorporated Groups, End-capped reagent in the tests where it is used.
A very finely divided silica gel (3-10 ¡lm). The partic1es are Silica Gel for Chromatography, Propoxybenzene, End-
based on silica, chemically modified with a reagent providing capped
a surface with chains having polar incorporated groups and A very finely divided (3 to 10 ¡lm) silica gel, chemically
terminating octyl groups. Furthermore, the packing material modified at the surface by the bonding of propoxybenzene
is end-capped. The particle size is indicated after üi.e name of groups. The particle size is indicated after the name of the
the reagent in the tests where it is used. reagent in the test where it is used.
Fine, white or almost white, homogeneous powder. Silica Gel for Chromatography, Propylsilyl
Silica Gel for Chromatography, Oxypropionitrilsilyl A very finely divided silica gel (3-10 ¡lm), chemically
A very finely divided silica gel chemically modified at the modified at the surface by the bonding of propylsilyl groups.
surface by the bonding of oxypropionitrilsilyl groups. The The particle size is indicated after the name of the reagent in
partic1e size is indicated after the name of the reagent in the the test where it is used.
tests where it is used. Silica Gel for Chromatography Rl, Nitrile
Silica gel for Chromatography, A very finely divided silica gel consisting of porous, spherical
Palmitamidopropylsilyl, End -capped particles with chernically bonded nitrile groups. The particle
A very finely divided (3 ro 10 ¡lm) silica gel, chemically size is indicated after the name of the reagent in tests where
modified at the surface by the bonding of palmitamidopropyl it is used.
groups and end-capped with acetamidopropyl groups. Fine, white or almost white, homogeneous powder,
The partic1e size is indicated after the na me of the reagent in practically insoluble in water and in ethanol (96%).
the tests where it is used. Silica Gel for Chromatography R2, Nitrile
Fine, white or almost white, homogeneous powder, Ultrapure silica gel, chemically modified at the surface by the
practically insoluble in water and in ethanol (96%) . introduction of cyanopropylsilyl groups. Less than 20 ppm of
Silica Gel for Chromatography, Phenyl metals. The partic1e size is indicated after the name of the
Liquid chromatographic reagent grade of commerce. reagent in the tests where it is used.
Fine white or almost white, homogenous powder, practically Fine, white or almost white, homogeneous powder,
insoluble in water and in ethanol (96%) . practically insoluble in water, in ethanol (96%) and in
Silica Gel for Chromatography Rl, Octadecylsilyl methylene chloride.
A very finely divided ultrapure silica gel, chemically modified Spheroidal silica, 8 nm; specific surface area, 180 mZper g;
at the surface by the bonding of octadecylsilyl groups. The carbon loading, 5.5%.
particle size, the pore size and the carbon loading are Silica Gel for Chromatography, Strong-anion-exchange
indicated after the name of the reagent in the tests where it is A very finely divided (3 ~lm-l0 ¡.tm) silica gel, chemically
used. Less than 20 ppm of metals. modified at the surface by the bonding of quatemary
Silica Gel for Chromatography R2, Octadecylsilyl ammonium groups. The particle size is indicated after the
A very finely divided (15 nm pore size) ultrapure silica gel, name of the reagent in the tests where it is used.
chemically modifed at the surface by the bonding of Fine, white or almost white, homogeneous powder,
octadecylsilyl groups (20% carbon load), optimised for the practically insoluble in water and in ethanol (96%).
analysis of polycyclic aroma tic hydrocarbons. The particle pH limit of use, 2 to 8.
size is indicated after the name of the reagent in the tests Silica Gel for Chromatography, Strong cation-
where it is used. exchange
Fine, white or almost white, homogeneous powder, A very finely divided (5 to 10 ¡.tm) silica gel, chemically
practically insoluble in water and in ethanol (96%). modified at the surface by the bonding of sulfonic acid
Silica Gel for Chromatography Rl, Octadecylsilyl, groups. The particle size is specified after the name of the
End-capped reagent in the tests where it is used.
A very finely divided (10 nm pore size) ultrapure silica gel, Silica Gel for Chromatography, Trimethylsilyl
chemically modified at the surface by the bonding of A very finely divided (3 to 10 ¡.tm) silica gel, chemically
octadecylsilyl groups (19% carbon load) . To minimise any modified at the surface by the bonding of trimethylsilyl
interaction with basic compounds it is carefully end-capped groups. The particle size is indicated after the name of the
to cover most of the remaining silanol groups. The particle reagent in the tests where it is used.
size is indicated after the name of the reagent in the tests
where it is used. It contains less than 20 ppm of metals. Fine, white or almost white, homogeneous powder,
practically insoluble in water and in ethanol (96%).
Silica Gel for Chromatography Rl, Octadecylsilyl,
End-capped, Base-deactivated Silica Gel for Size-exclusion Chromatography
A very finely divided (3-10 ¡.tm) silica gel pre-treated before Chromatographic reagent grade of commerce.
the bonding of octadecylsilyl groups by washing and A very finely divided silica gel (10 ¡.tm) with a very
hydrolysing most of the superficial siloxane bridges. hydrophilic surface. The average diameter of the pores is
To further minimise any interaction with basic compounds it about 30 nm. It is compatible with aqueous solutions
is carefully end-capped to cover most of the remaining silanol between pH 2 and 8 and with organic solvents. It is suitable
groups. The particle size is indicated after the name of the for the separation of proteins with relative molecular mas ses
reagent in the test where it is used. of 1 x 10 3 to 3 x 10 5 .
Fine, white or almost white, homogeneous powder, Silica Gel F 254
practically insoluble in water and in ethanol (96%) . A fine, white, homogeneous powder of an average particle
Silica Gél for Chromatography Rl, Octylsilyl size of about 15 ¡.tm containing a suitable binding agent and
A very finely divided (3 ¡.tm-l O ~lm) silica gel, chemically about 1.5% of a florescent indicator having a maximum
modified at the surface by the bonding of octylsilyl and intensity at 254 nm. It complies with the following
methyl groups (double bonded phase). The particle size is requirement.
indicated after the name of the reagent in the tests where it is Fluorescence Carry out the method for thin-layer
used. chromatography using a mixture of 10 volumes of anhydrous
Fine, white or almost white, homogeneous powder, formic acid and 90 volumes of propan-2-ol as the mobile
practically insoluble in water and in ethanol (96%). phase, but allowing the solvent front to ascend 10 cm aboye
the line of application. Apply separately to the plate 10
Silica Gel for Chromatography R2, Octylsilyl quantities from I to 10 uL of a 0.1 % w/v solution of benzoic
Ultrapure very finely divided (10 nm pore size) silica gel, acid in the mobile phase. After removal of the plate, dry it in
chemically modified at !he surface by the bonding of a current of warm air and examine under ultraviolet light
octylsilyl groups (19% carbon load). Less than 20 ppm of (254 nm). The benzoic acid appears as dark spots on a
metals. fluorescent background in the upper third of the
Silica Gel for Chromatography R3, Octylsilyl chromatogram at levels of 2 ¡.tg and greater.
A very finely divided ultrapure silica gel, chemically modified Silica Gel G (J 12926-00-8)
at the surface by the bonding of octylsilyl groups and Contains about 13% of calcium sulfate hemihydrate.
sterically protected with branched hydrocarbons at the Fine, white or almost white, homogeneous powder with a
silanes. The particle size is indicated after the name of the particle size of about 15 ¡.tm.
reagent in the tests where it is used. Calcium sulfate content Place 0.25 g in a ground-glass
Silica Gel for Chromatography Rl, Phenylsilyl stoppered flask, add 3 mL of dilute hydrochloric aCld and
A very finely divided silica gel (5 ¡.tm), chemically modified at 100 mL of water and shake vigorously for 30 minutes. Filter
the surface by the bonding of phenyl groups. The particle through a sintered-glass filter (2. 1.2) and wash the residue.
size is indicated after the name of the reagent in the tests Carry out on the combined filtrate and washings the
where it is used. complexometric assay of calcium (2.5.11).
1 mL of 0.1 M sodium edetate is equivalent to 14.51 mg of
CaS04,lhH zO.
pH (2.2.3) Shake 1 g for 5 minutes with 10 mL of carbon with three quantities, each of 10 mL of ehloroform. Dry the
dioxide-free water. The pH of the suspension is about 7. combined chloroform extracts over anhydrous sodium sulfate,
Silica Gel GFZ54 (112926-00-8) filter and evaporate to dryness on a water-bath. Dissolve the
residue in 50 mL of eh/oroform. Examine by thin-Iayer
Contains about 13% of calcium sulfate hemihydrate and
chromatography (2.2.27), using silanised silica gel HF2s4 as
about 1.5% of a fiuorescent indicator having an optimal
the coating substance. Apply to the plate at each of three
intensity at 254 nm.
separate points 10 ¡.tL of the chloroformic solution. Develop
Fine, white or almost white, homogeneous powder with a over a path of 14 cm with a mixture of 10 volumes of glacial
particIe size of about 15 ¡.tm. aeetie acid, 25 volumes of water and 65 volumes of dioxan .
Calciurn sulfate content Determine by the method Dry the plate at 120° for 30 minutes. AlIow to cool, spray
prescribed for siliea gel G. with a 35 gIL solution of phosphomolybdie acid in 2-propanol
pH (2.2.3) Complies with the test prescribed for siliea gel and heat at 150° until the spots become visible. Treat the
G. plate with arnmonia vapour until the background is white.
Fluorescence Thin-Iayer chromatography (2.2.27) using The chromatograms show four cIearly separated, well-
silica gel GF254 as the coating substance. Apply separately to defined spots.
the plate at ten points increasing volumes from 1 ¡.tL to 10 Silica Gel OC for Chiral Separations A very finely
¡.tL of a 1 gIL solution of benzoie acid in a mixture of divided silica gel for chromatography (5 ¡.tm) coated with the
10 volumes of anhydrous formie acid and 90 volumes of 2- following derivative:
propano!. Develop over a path of 10 cm with the same
mixture of solvents. After evaporating the solvents examine
the chromatogram in ultraviolet light at 254 nm.
The benzoic acid appears as dark spots on a fiuorescent
background in the upper third of the chromatogram for
quantities of 2 ¡.tg and greater.
Silica Gel H (112926-00-8)
Fine, white or almost white, homogeneous powder with a
particIe size of about 15 ¡.tm. Silica Gel OD for Chiral Separations
pH (2.2.3) Complies with the test prescribed for siliea gel A very finely divided silica gel for chromatography (5 ¡.tm)
G. coated with the following derivative.
Silica Gel H, Silanised
Preparation 01 a thin layer See silanised siliea gel HF254 .
A fine, white or almost white homogeneous powder which, OR
after being shaken with water, fioats on the surface because R =

of its water-repellent properties.
Chrornatographic separation Complies with the test
prescribed for silanised siliea gel HF254 •
Silica Gel HF z54 Silica Gel 01 for Chiral Separations
Contains about 1.5% of a fiuorescent indicator having an A very finely divided silica gel for chromatography consisting
optimal intensity at 254 nm. of spherical particIes coated with cellulose
Fine, white or almost white, homogeneous powder with a tris(4-methylbenzoate). The particIe size is indicated after the
particIe size of about 15 ¡.tm. name of the reagent in the test where it is used.
pH Complies with the test prescribed for si/iea gel G. Silicotungstic Acid Dodecatungstosilicic acid;
Fluorescence Complies with the test prescribed for siliea H4SiW1 2040' + aq (11130-20-4)
gel GF254 . White or yellowish-white crystals, deliquescent, very soluble
Silica Gel HF z54 , Silanised in water and in ethanol (96%).
Contains about 1.5% of a fiuorescent indicator having an Store in an airtight container.
optimal intensity at 254 nm. Silicristin (2R,3R)-3,5, 7-Trihydroxy-2-[(2R,3S)-7-
Fine, white or almost white, homogeneous powder which, hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxyrnethyl-
after shaking with water, fioats on the surface because of its 2,3-dihydro-l-benzofuran-5-yl] chroman-4-one;
water-repellent properties. C2sH2201O = 482.4 (33889-69-9)
Preparation 01 a thin layer Vigorously shake 30 g for White or yellowish powder, practically insoluble in water,
2 minutes with 60 mL of a mixture of 1 volume of methanol soluble in acetone and in methanol.
and 2 volumes of water. Coat carefully cIeaned plates with a Silidianin (3R,3aR,6R, 7 aR,8R)-7 a-Hydroxy-8-( 4-hydroxy-
layer 0.25 mm thick using a spreading device. AlIow the 3-methoxyphenyl)-4- [(2R, 3R)- 3,5, 7 -trihydroxy-4-
coated plates to dry in air and then heat in an oven at 100° oxochroman-2-yl]-2,3,3a,7 a-tetrahydro-3,6-methano-l-
to 105° for 30 minutes. benzofuran-7 (6aH)-one; C 2sH 22 0 10 = 482.4 (29782-68-1 )
Chrornatographic separation Introduce 0.1 g each of White or yellowish powder, practically insoluble in water,
methyl laura te, methyl myristate, methyl palmitate and methyl soluble in acetone and in methanol.
stearate into a 250 mL conical fiask. Add 40 mL of aleoho/ie Silver Diethyldithiocarbamate Diethyldithiocarbamic
potassium hydroxide solution and heat under a refiux condenser acid silver salt; C SH IO AgNS2 = 256.1 (1470-61-7)
on a water-bath for 1 hour. AlIow to cool, transfer the
Pale-yellow or greyish-yellow powder, practically insoluble in
solution to a separating funnel by means of 100 mL of water,
water, soluble in pyridine.
acidify (pH 2 to 3) with dilute hydroehlorie aeid and shake
It may be prepared as follows. Dissolve 1.7 g of silver nitrare Assay Gas chromatography (2.2.28), as prescribed in the
in 100 mL of water. Separately dissolve 2.3 g of sodium monograph Phytosterol (1911).
diethyldithiocarbamate in 100 mL of water. Cool both Test solution Dissolve 0.100 g of the substance to be
solutions to 10°, then mix and while stirring collect the examined in tetrahydrofuran and dilute to 10.0 mL with the
yellow precipitate on a sintered-glass fi1ter (2.1.2) and wash same solvent. Introduce 100 ~L of this solution into a
with 200 mL of cold water. Dry the precipitate in vacuo for suitable 3 mL f1ask and evaporate to dryness under nitrogen.
2-3 hours. To the residue add 100 ~lL of a freshly prepared mixture of
Silver diethyldithiocarbamate may be used provided it has 50 ~LL of 1-merhyli111idazole and l.0 mL of heptafiuoro-N-
not changed in colour or developed a strong odour. methyl-N-(trimethylsilyl)butanamide. Close the f1ask tightly and
Silver Manganese Paper Immerse strips of slow filter heat at 100° for 15 minutes. AlIow to cool.
paper into a solution containing 8.5 giL of manganese sulfate Injection 1 ~L of the test solution.
and 8.5 giL of silver nitrate. Maintain for a few minutes and Sodium Na = 22.99 (7440-23-5)
allow to dry over diphosphorus pentoxide protected from acid A metal whose freshly cut surface is bright silver-grey.
and alkaline vapours. It rapidly tamishes in contact with air and is oxidised
Silver Nitrate AgN0 3 = 169 .9 (7761-88-8) completely to sodium hydroxide and con verted to sodium
Of the British Pharmacopoeia. carbonate . It reacts violently with water, yielding hydrogen
Silver Nitrate Reagent To a mixture of 3 mL of and a solution of sodium hydroxide ; soluble in anhydrous
concentrated ammonia and 40 mL of 1 M sodium hydroxide, methanol, yielding hydrogen and a solution of sodium
add 8 mL of a 200 gIL solution of silver nitrate, dropwise, methoxide; practically insoluble in light petroleum.
with stirring. Dilute to 200 mL with water. Store under light petroleum or liquid paraffin.
Silver Nitrate Solution Sodium Acetate C 2 H 30 2Na,3H 20 = 136.1 (6 131-90-4)
A freshly prepared 5.0% w/v solution of silver nitrate. Sodium Acetate Trihydrate of the British Pharmacopoeia.
Store protected from light. Sodium Acetate, Anhydrous
Silver Nitrate Solution, Ammoniacal Dissolve 2.5 g of C 2 H 30 2Na = 82.0 (127-09-3)
si/ver nitrate in 80 mL of water and add dilute ammonia R1 Colourless crystals or granules, very soluble in water,
dropwise until the precipitate has dissolved. Dilute to sparingly soluble in ethanol (96%).
100 mL with water. Prepare irnmediately before use. Loss on drying (2.2.32) Not more than 2.0%, determined by
Silver Nitrate Solution in Pyridine An 8.5% w/v drying in an oven at 105°.
solution in pyridine. Sodium Arsenite Sodium metaarsenite;
Store protected from light. NaAs0 2 = 129.9 (7784-46-5)
Silver Nitrate Solution Rl A 4.25% w/v solution. Analytical reagent grade of commerce.
Store protected from light. Sodium arsenite solution
Silver Nitrate Solution R2 A l.7 % w/v solution. Dissolve 5.0 g of sodiu111 arsenite in 30 mL of 1 M sodiu111
Store protected from light. hydroxide. Cool to 0° and add, while stirring, 65 mL of dilute
hydrochloric acid.
Silver Oxide Disilver oxide; Ag20 = 23l.7 (20667-12-3)
Brownish-black powder, practically insoluble in water and in Sodium Ascorbate Solution (134-03-2)
ethanol (96%), freely soluble in dilute nitric acid and in Dissolve 3.5 g of ascorbic acid in 20 mL of 1 M sodium
arnmonia. hydroxide. Prepare immediately before use. .
Store protected from light. Sodium Azide NaN3 = 65.0 (26628-22-8)
Sinensetin 3',4' ,5,6, 7-Pentamethoxyflavone; White or almost white, crystalline powder or crystals, freely
CZOH2007 = 372.4 (2306-27-6) soluble in water, slightly soluble in ethanol (96%).
White or almost white, crystalline powder, practically Sodium Bicarbonate See Sodiu111 hydrogen carbonate.
insoluble in water, soluble in ethanol (96%). Sodium Bismuthate NaBi0 3 = 280.0 (12232-99-4)
Melting point, about 177°. Content, minimum 85.0%.
Absorbance (2.2.25). A solution in methanol shows 3 Yellow or yellowish-brown powder, slowly decomposing
absorption maxima, at 243 nm, 268 nm and 330 nm. when moist or at a high temperature, practically insoluble in
Assay Liquid chromatography (2.2.29) as prescribed in the cold water.
monograph Java tea (1229). Assay Suspend 0.200 g in lO mL of a 200 gIL solution of
Content, minimum 95%, ca1culated by the normalisation potassiu111 iodide and add 20 mL of dilute sulfuric acid. Using
procedure. I mL of starch solurion as indicator, titrate with 0.1 M sodiu111
Sinomenine 7,8-Didehydro-4-hydroxy-3, 7-dimethoxy-l 7- thiosulfate until an orange colour is obtained.
methyl-9cx, l3cx, l4cx-morphinan-6-one; Cucoline; 1 mL of 0.1 M sodium rhiosulfate is equivalent to 14.00 mg of
C19H23N04 = 329.4 (115-53-7) NaBi0 3·
Sitostanol Dihydro-~-sitosterol; Sodium Bromide NaBr = 102.9 (7647-15-6)
C 29 H 52 0 = 416.7 (19466-47-8) Of the British Pharmacopoeia.
Content, minimum 95.0%. Sodium Butanesulfonate l-Butanesulfonic acid sodium
~-Sitosterol 22,23-Dihydrostigmasterol, stigmast-5-en- 3~- salt, 1-butanesulfonic acid sodium salt, sodium
01; C 29 H so O = 414.7 (83-46-5) butanesulfonate; C4H9Na03S = 160.2 (2386-54-1)
White or almost white powder, practically insoluble in water, White or almost white, crystalline powder, soluble in water.
sparingly soluble in tetrahydrofuran. Melting point, greater than 300°.
Content, minimum 75.0% mlm (dried substance).
2014 Appendix lA V-Al19
Sodium Calcium Edetate (62-33-9) Sodium Deoxyribonucleate About 85% has a relative
Of the British Pharmacopoeia. molecular mas s of 2 x 10 7 or greater. (73049-39-5)
Sodium Carbonate Na2C03,10H20 = 286.2 (6132-02-1) White or almost white, fibrous preparation obtained from calf
thymus.
Sodium Carbonate Decahydrate of the British
Pharmacopoeia. Testfor suitability Dissolve 10 mg in imídazole buffer
solutíon pH 6.5 and dilute ro 10.0 mL with the same buffer
Sodium Carbonate, Anhydrous Disodium carbonate;
solution (solution A) . Dilute 2.0 mL of solution A ro
Na2C03 = 106.0 (497-19-8)
50.0 mL with ímídazole buffer solution pH 6.5 .
White or almost white powder, hygroscopic, freeJy soluble in The absorbance (2.2.25) of the solution, measured at
water. 260 nm, is 0.4 to 0.8.
0
When heated ro about 300 ir loses not more than 1% of its To 0.5 mL of solution A add 0.5 mL of imídazole buffer
mass. soluríon pH 6.5 and 3 mL of perchloric acid (25 gIL HCI0 4).
Srore in an airtight container. A precipitate is formed. Centrifuge. The absorbance of the
Sodium Carbonate Monohydrate supernatant liquid, measured at 260 nm using a mixture of
Na2C03,H20 = 124.0 (5968-11-6) 1 mL of imídazole buffer soluríon pH 6.5 and 3 mL of
Of the British Pharmacopoeia. perchloric acid (25 gIL HCI0 4) as compensation liquid, is
not greater than 0.3.
Sodium Carbonate Solution A 10.6% w/v solution of
anhydrous sodium carbonate. In each of two tubes, place 0.5 mL of solution A and 0.5 mL
of a solution of a reference preparation of streprodomase
Sodium Carbonate Solution, Dilute A 10% w/v solution
containing 10 IU/mL in imidazole buffer solution pH 6.5.
of sodium carbonate.
To one tube add immediately 3 mL of perchloric acid
Sodium Carbonate Solution Rl A 2% w/v solution of (25 giL HCI0 4). A precipitate is formed. Centrifuge and
anhydrous sodíum carbonate in O.lM sodíum hydroxíde. collect the supematant liquid A. Heat the other tube at 37°
Sodium Carbonate Solution R2 A 4% w/v solution of for 15 minutes and add 3 mL of perchloric acid (25 giL
anhydrous sodíum carbonate in 0.2 M sodium hydroxide. HCI0 4). Centrifuge and collect the supernatant liquid B.
Sodium Cetostearyl Sulfate Sodium cetostearyl sulphate The absorbance of supernatant liquid B, measured at
Of the British Pharmacopoeia. 260 nm with reference ro supernatant liquid A is not less
than 0.15.
Sodium Chloride NaCI = 58.44 (7647-14-5)
Sodium Diethyldithiocarbamate
C SH lO NNaS2>3H 20 = 225.3 (20624-25-3)
Sodium Chloride Solution A 20% w/w solution of
White or almost white or colourless crystals, freely soluble in
sodíum chloride.
water, soluble in ethanol (96%). The aqueous solution is
Sodium Chloride Solution, Saturated Mix 1 part of colourless.
sodíum chloride with 2 parts of water, shake from time to time
Sodium Diethyldithiocarbamate Solution A 0.1 % w/v
and allow ro stand. Before use, decant the solution from any
solution of sodium diethyldithiocarbamate in water. •
undissolved substance and filter, if necessary.
Prepare immediately before use.
Sodium Cholate Cholic acid sodium salt hydrate;
C24H39NaOs = 430.5 (73163-53-8) Sodium Dihydrogen Orthophosphate Sodium
dihydrogen phosphate, sodium dihydrogen phosphate
dihydrate; NaH 2P0 4,2H zO = 156.0 (10028-24-7)
[a)j3l, +31.3 (0.5% w/v in water).
Sodium Dihydrogen Phosphate Dihydrate of the British
Sodium Citrate Trisodium citrate; Pharmacopoeia.
C6HsNa307,2H20 = 294.1 (6132-04-3)
Sodium Dihydrogen Orthophosphate, Anhydrous See
Of the British Pharmacopoeia. anhydrous sodium dihydrogen phosphate.
Sodium Cobaltinitrite Sodium hexanitrirocobaltat~(III); Sodium Dihydrogen Orthophosphate Monohydrate
Na3[CO(N02)]6 = 403.9 (13600-98-1) See sodium dihydrogen phosphate monohydrate.
Orange-yellow powder, freely soluble in water, slightly Sodium Dihydrogen Phosphate See sodium d¡hyhydrogen
soluble in ethanol (96%). orthophosphate
Sodium Cobaltinitrite Solution A 10% w/v solution. Sodium Dihydrogen Phosphate, Anhydrous NaH 2 P0 4
Prepare immediately before use. = 120.0 (7558-80-7)
Sodium Decanesulfonate Sodium decanesulfonate; White or almost white powder, hygroscopic.
ClOH2lNa03S = 244 .3 (13419-61-9)
Srore in an airtight container.
CrystalJine powder or flakes, white or almost white, freeJy
Sodium Dihydrogen Phosphate Monohydrate
soluble in water, soluble in methanol.
NaH 2 P0 4,H 20 = 138.0 (10049-21-5)
Sodium Decyl Sulfate Sodium decyl sulfate;
White or almost white, slightly deliquescent crystals or
C lO H2JNa04S = 260.3 (142-87-0)
granules, freeJy soluble in water, practically insoluble in
Content, minimum 95.0%. ethanol (96%).
White or almost white powder, freely soluble in water. Store in an airtight container.
Sodium Deoxycholate Deoxycholic acid, sodium salt; Sodium Diocty1 Sulfosuccinate Sodium
sodium 30:, 120:-dihydroxy-5 ~-cholan-24-oate; 1,4-bis [(2-ethylhexyl)oxy]-1 ,4-dioxobutane-2-sulfonate,
C24H39Na04 = 414.6 (302-95-4) 1,4-bis(2-ethylhexyl) sulfobutanedioate sodium salt;
General reagent grade of commerce. C2oH37Na07S = 444 .6 (577-11-7)
White or almost white, waxy solido
Sodium Dithionite Na2S204 = 174.1 (7775-14-6) Sodium Hexanesulfonate Monohydrate for Ion-pair
White or greyish-white, crystalline powder, oxidises in air, Chromatography
very soluble in water, slight1y soluble in ethanol (96%). C6H13Na03S,H20 = 206.2 (207300-91-2)
Store in an airtight container. Content, minimum 99.0%.
Sodium Dodecyl Sulfate Sodium dodecyl sulphate, Sodium Hydrogen Carbonate Sodium bicarbonate;
sodium lauryl sulfate, sodium lauryl sulphate, sodium NaHC0 3 = 84.01 (144-55-8)
laurilsulfate; Cl2H2SNa04S = 288.4 (151-21-3) Sodium Bicarbonate of the British Pharmacopoeia.
Of the British Pharmacopoeia. Sodium Hydrogen Carbonate Solution A 4.2% w/v
Content, minimum 99.0%. solution.
Sodium Edetate (6381-92-6) Disodium Edetate of the Sodium Hydrogen Sulfate Sodium bisulfate, sodium
British Pharmacopoeia. bisulphate, sodium hydrogen sulphate;
NaHS0 4 = 120.1 (7681-38-1)
Sodium Fluoresceinate CI 45350, Fluorescein sodium,
disodium 2-(3-oxo-6-oxido-3H-xanthen-9-yl)benzoate; Freely soluble in water, very soluble in boiling water. It
C2oHIONa20S = 376.3 (518-47-8) decomposes in ethanol (96%) into sodium sulfate and free
Orange-red powder, freely soluble in water. Aqueous sulfuric acid.
solutions display an intense yellowish-green fluorescence. Melting point, about 315°.
Sodium Fluoride NaF = 41.99 (7681-49-4) Sodium Hydrogensulfite Sodium hydrogensulphite;
Of the British Pharmacopoeia. NaHS0 3 = 104.1 (7631-90-5)
When used in the Assay of preparations containing Sodium White or almost white, crystalline powder, freely soluble in
Fluoride, use a grade containing not less than 99.9% ofNaF. water, sparingly soluble in ethanol (96%).
Sodium Formate Sodium methanoate; On exposure to air, sorne sulfur dioxide is lost and the
HC0 2Na = 68.0 (141-53-7) substance is gradually oxidised to sulfate.
White or almost white, crystalline powder or deliquescent Sodium Hydroxide NaOH = 40.00 (1310-73-2)
granules, soluble in water and in glycerol, slight1y soluble in Of the British Pharmacopoeia.
ethanol (96%). 2M Sodium hydroxide
Melting point, about 253°. Dissolve 84 g of sodium hydroxide in earbon dioxide-free water
Sodium Glucuronate D-Glucuronic acid, somum salt; and dilute to 1000.0 mL with the same solvent.
C6H9Na07,H20 = 234.1 Sodium Hydroxide, Ethanolic Solutions of the requisite
[a]f,O, about +2 1.5, determined on a 2% w/v solution. molarity may be obtained by dissolving the appropriate
Sodium Glycocholate Sodium [(3,7, 12-trihydroxy-5- amount of sodium hydroxide in sufficient ethanol (96%) to
cholan-24-oyl)amino ]acetate dihydrate; N-[(3,5,7,12)-3, 7,12- produce 1000 mL.
Trihydroxy-24-oxocholan-24-yl]glycine monosodium salt Sodium Hydroxide, Methanolic Solutions of the
dihydrate; C26H42NNa06,H20 = 523.6 (207300-80-9) requisite molarity may be obtained by dissolving the
Content, minimum 97% of C26H42NNa06,2H20. appropriate amount of sodium hydroxide in sufficient methanol
to produce 1000 mL.
Sodium Heptanesulfonate l-Heptanesulfonic acid
sodium salt; l-heptanesulphonic acid sodium salt; sodium Sodium Hydroxide Solution Dissolve 20.0 g of sodium
heptanesulphonate; C7H1SNa03S ~ 202.3 (22767-50-6) hydroxide in water and dilute to 100.0 mL with the same
White or almost white, crystalline mas s, freely soluble in solvent. Verify the concentration by titration with 1 M
water, soluble in methano!. hydroehloric acid, using methyl orange solution as indicator, and
Sodium Heptanesulfonate Monohydrate adjust if necessary to 200 gIL.
l-Heptanesulfonic acid sodium salt monohydrate; 1- Sodium Hydroxide Solution, Carbonate-free
heptanesulphonic acid sodium salt monohydrate; sodium Dissolve sodium hydroxide in carbon dioxide-free water to give a
heptanesulphonate monohydrate; concentration of 50% w/v and allow to stand. Decant the
C7H1SNa03S,H20 = 220.3 clear supernatant liquid, taking precautions to avoid the
Content, minimum 96% (anhydrous substance). introduction of carbon dioxide.
White or almost white, crystalline powder, soluble in water, Sodium Hydroxide Solution, Dilute Dissolve 8.5 g of
very slightly soluble in anhydrous ethano!. sodium hydroxide in water and dilute to 100 mL with the
Water (2.5.12) MaxÍmum 8%, determined on 0.300 g. same solvento
Assay Dissolve 0.150 g in 50 mL of anhydrous aeetie acid. Sodium Hydroxide Solution, Methanolic Dissolve
Titrate with 0.1 M perehlorie acid, determining the end-point 40 mg of sodium hydroxide in 50 mL of water. Cool and add
potentiometrically (2.2.20). 50 mL of methanol.
1 mL of 0.1 M perehlorie aeid is equivalent to 20.22 mg of Sodium Hydroxide Solution Rl, Methanolic Dissolve
C7H1 SNa03S, 200 mg of sodium hydroxide in 50 mL of water. Cool and add
Sodium Hexanesulfonate Hexanesulfonic acid sodium 50 mL of methanol.
salt, hexanesulphonic acid sodium salt, sodium Sodium Hydroxide Solution, Strong Dissolve 42 g of
hexanesulphonate; C6H13Na03S = 188.2 (2832-45-3) sodium hydroxide in water and dilute to 100 mL with the
White or almost white powder, freely soluble in water. same solvent.
Sodium Hexanesulfonate Monohydrate Hexanesulfonic Sodium 2-Hydroxybutyrate Sodium (2RS)-2-
acid sodium salt monohydrate, hexanesulfonic acid sodium hydroxybutanoate; C4H7Na03 = 126.1 (19054-57-0)
salt monohydrate, sodium hexanesulfonate monohydrate; General reagent grade of commerce.
C6H13Na03S,H20 = 206.2 (207300-91 -2)
White or almost white powder, soluble in water.
2014 Appendix lA V-A121
Sodium Hypobromite Solution In a bath of iced water Sodium Methanesulfonate Methanesulfonic acid, sodium
mix 20 mL of strong sodium hy droxide solution and 500 mL of salt; methanesulphonic acid, sodium salt; sodium
water, add 5 mL of bromine solution and stir gently until methanesulphonate; CH 3S03Na = 118.1 (2386-57-4)
solution is complete. Prepare immediately before use . White or almost white, crystalline powder, hygroscopic.
Sodium Hypochlorite Solution Store in an airtight container.
General reagent grade of commerce containing lOto Sodium Molybdate Disodium molybdate dihydrate;
14% w /v of available chlorine. Na2Mo04,2H20 = 242 .0 (10102-40-6)
Sodium Hypochlorite Solution (3% el) Strong sodium White or almost white, crystalline powder or colourless
hypochlorite solution crystals, freely soluble in water.
See sodiwn hypochlorite solution, strong. Sodium Naphthoquinonesulfonate Sodium
Sodium Hypochlorite Solution, Dilute Dilute 35 mL of 1,2-naphthoquinone-4-sulfonate;
sodium hypochlorite solution to 100 mL with water immediately CIOHsNaOsS = 260 .2 (521-24-4)
before use. Yellow or orange-yellow, crystalline powder, freely soluble in
The solution contains approximately 3.5% w/v of available water, practically insoluble in ethanol (96%).
chlorine. Sodium 1,2-Naphthoquinone-4-sulfonate See Sodium
Sodium Hypochlorite Solution, Strong naphthoquinonesulfonate.
Content, 2.5% w/v to 3.0% w/v of active chlorine. Sodium Nitrate NaN0 3 = 85.0 (7631-99-4)
Yellowish liquid with an alkaline reaction. White or almost white powder or granules or colourless,
Assay Introduce into a ftask, successively, 50 mL of water, transparent crystals, deliquescent in moist air, freely soluble
1 g of potassium iodide and 12.5 mL of di/ute acetic acid. in water, slightly soluble in ethanol (96 %).
Dilute 10.0 mL ofthe substance 10 be examined to Store in an airtight container.
100.0 mL with water. Introduce 10.0 mL of this solution Sodium Nitrite NaN0 2 = 69.0 (7632-00-0)
into the ftask and titrate with 0.1 M sodium thiosulfate, using Content, minimum 97.0% .
1 mL of starch solution as indicator.
White or almost white, granular powder or a slightly yellow,
1 mL of 0.1 M sodium thiosulfate is equivalent to 3.546 mg of crystalline powder, freely soluble in water.
active chlorine.
Assay Dissolve 0.100 g in 50mL of water. Add 50.0 mL of
Store protected from light. O. 02 M potassium permanganate and 15 mL of dilute sulphuric
Sodium Hypophosphite Sodium phosphinate acid. Add 3 g of potassium iodide. Titrate with 0.1 M sodium
monohydrate; NaH 2P0 2,H 20 = 106.0 (10039-56-2) thiosulfate, using 1.0 mL of starch solution added towards the
White or almost white, crystalline powder or colourless end of the titration as indicator.
crystals, hygroscopic, freely soluble in water, soluble in 1 mL of 0.02 M potassium pennanganate is equivalent to
ethanol (96%). 3.450 mg ofNaN0 2.
Store in an airtight container. Sodium Nitrite Solution A 10% w /v solution. Prepare
Sodium Iodide NaI = 149 .9 (7681-82-5) immediately before use.
Of the British Pharmacopoeia . Sodium Nitroprusside Sodium pentacyano-
Sodium Iodobismuthate Solution Boil for a few minutes nitrosylferrate(m) dihydrate;
a mixture of 2.6 g of bismuth oxycarbonate, 7.0 g of sodium Na2[Fe(CN)s(NO)],2H20 = 298.0 (13755-38-9)
iodide and 25 mL of glacial acetic acid. Allow to stand for 12 Reddish-brown powder or crystals, freely soluble in water,
hours and filter, if necessary, through sintered glass. To slightly soluble in ethanol (96%) .
20 mL of the filtrate add 80 mL of ethyl acetate (solution A) . Sodium Nitroprusside-earbonate Solution Dissolve 1 g
Immediately before use, mix 2 mL of solution A, 20 mL of . of sodium nitroprusside and 1 g of anhydrous sodium carbonate
glacial acetic acid and 40 mL of ethyl acerate. in sufficient water to produce 100 mL.
For use in co=ection with thin-Iayer chromatography the Sodium Octanesulfonate Octanesulfonic acid sodium
sensitivity may be increased by spraying first with this salt; octanesulphonic acid sodium salt; sodium
solution and then with sulfuric acid (0.2%). octanesulphonate; CsH17Na03S = 216.3 (5324-84-5)
Solution A should be kept in a well-closed container. Content, minimum 98 .0% .
Sodium Laurilsulfate See sodium dodecyl sulfate. White or almost white, crystalline powder or ftakes, freely
Sodium Lauryl Sulfate See sodium dodecyl sulfate. soluble in water, soluble in methanol.
Sodium Laurylsulfonate for ehromatography Sodium Absorbance (2.2.25) Maximum 0.10, determined at
laurylsulphonate for chromatography; 200 nm and maximum 0.01, determined at 250 nm using a
Cl2H2SNa03S = 272.4 (2386-53-0) 54 giL solution.
White or almost white powder or crystals, freely soluble in Sodium Octanesulfonate Monohydrate Sodium
water. octanesulphonate monohydrate;
Absorbance (2.2.25), determined in water: about 0.05 at CsH17Na03S,H20 = 234.3 (207596-29-0)
210 nm; about 0.03 at 220 nm; about 0.02 at 230 nm; White or almost white powder.
about 0.02 at 500 nm. Sodium Octyl Sulfate 4-0ctyl sulfate sodium salt, 4-octyl
Sodium Metabisulfite Sodium metabisulphite, sodium sulphate sodium salt, sodium octyl sulphate;
pyrosulfite, sodium pyrosulphite; CsH 17Na04S = 232 .3 (142-31-4)
Na2S20S = 190.1 (7681-57-4) White or almost white, crystalline powder or ftakes, freely
Of the British Pharmacopoeia . soluble in water, soluble in methanol.
Sodium Oxalate C2Na204 = 134.0 (62-76-0) Loss on drying (2.2.32) Maximum 0.5%, determined by
White or almost white, crystalline powder, soluble in water, drying in an oven at 1300
•
practically insoluble in ethanol (96%). Sodium sulfate, anhydrous Rl

Sodium Pentanesulfonate l-Pentanesulfonic acid sodium Complies with the requirements prescribed for anhydrous
salt, l-pentanesulphonic acid sodium salt, sodium sodium sulfate with rhe folIowing maximum contents.
pentanesulphonate; C5HllNa03S = 174.2 (22767-49-3) CI, 20 ppm; Pb, 10 ppm; As, 3 ppm; Ca, 50 ppm; Fe,
White or almost white, crystalline solid, soluble in water. 10 ppm; Mg, 10 ppm.
Sodium Pentanesulfonate Monohydrate 1- Sodium Sulfate Decahydrate
Pentanesulfonic acid sodium salt monohydrate; 1- Na zS04,10H zO = 322.2 (7727- 73-3)
pentanesulphonic acid sodium salt monohydrate; sodium Sodium Sulfate of the British Pharmacopoeia.
pentanesulphonate monohydrate;
Sodium Sulfide Disodium sulfide nonahydrate, sodium
C5HllNa03S,H20 = 192.2 (207605-40-1) sulphide; Na zS,9H 20 = 240.2 (1313-84-4)
White or almost white crystalline solid, soluble in water. Colourless, rapidly yellowing crystals, deliquescent, very
Sodium Pentanesulfonate Monohydrate Rl Sodium soluble in water.
pentanesulphonate monohydrate Rl; Store in an airtight container.
C5HllNa03,HzO = 192.2 (207605-40-1)
Sodium Sulfide Solution Sodium sulphide solution
Content, minimum 99% of C5HllN03,HzO.
Dissolve 12 g of sodium sulfide with heating in 45 mL of a
Sodium Perchlorate NaCI0 4,H 20 = 140.5 (7791-07-3)
mixture of 10 volumes of water and 29 volumes of glycerol
Content, minimum 99.0% ofNaCI0 4,H 20. (85%), allow to cool and dilute to 100 mL with the same
White or almost white, deliquescent crystals, very soluble in mixture of solvents.
water. The solution should be colourless.
Store in a well-c1osed container. Sodium Sulfide Solution Rl Sodium sulphide
Sodium Periodate Sodium metaperiodate; solution Rl
NaI0 4 = 213 .9 (7790-28-5) Prepare by one of the folIowing methods .
Content, minimum 99.0%. - Dissolve 5 g of sodium sulfide in a mixture of 10 mL of
White or almost white, crystalline powder or crystals, soluble water and 30 mL of glycerol.
in water and in mineral acids. - Dissolve 5 g of sodium hydroxide in a mixture of 30 mL of
Sodium Periodate Solution Dissolve 1.07 g of sodium water and 90 mL of glycerol. Divide the solution into 2 equal
periodate in water, add 5 mL of dilute sulfuric acid and dilute portions. Saturate 1 portion with hydrogen sulfide, with
to 100.0 mL with water. Use a freshly prepared solution. cooling. Mix the 2 portions.
Sodium Phosphite See Sodium phosphite pentahydrate. Store in a welI-filIed container, protected from Iight; use
Sodium Phosphite Pentahydrate within 3 months.
Na2HP03,5HzO = 216.0 (13517-23-2) Sodium Sulfite Sodium sulphite;
White or almost white, crystalline powder, hygroscopic, freely Na ZS03,7H zO = 252.2 (10102-15-5)
soluble in water. Sodium Sulfite Heptahydrate of the British Pharmacopoeia.
Store in an airtight container. Sodium Sulfite, Anhydrous Sodium sulphite, anhydrous;
Sodium Picrate Solution, Alkaline Mix 20 mL of picric Na ZS03 = 126.0 (7757-83- 7)
acid solution and 10 mL of a 5% w/v solution of sodium Of the British Pharmacopoeia.
hydroxide and dilute to 100 mL with water. Sodium Tartrate Disodium (2R,3R)-2,3-
Use within 2 days. dihydroxybutanedioate dehydrate, sodium (+ )-tartrate;
Sodium Potassium Tartrate Potassium sodium (+)- C4H4NazOó,2H20 = 230.1 (6106-24-7)
tartrate; C 4H 4KNaO ó,4H zO = 282.2 (6381-59-5) White or almost white crystals or granules, very soluble in
Colourless, prismatic crystals, very soluble in water. water, practically insoluble in ethanol (96%) .
Sodium Pyrophosphate Tetrasodium diphosphate Sodium (+)-Tartrate See sodium tartrate.
decahydrate; Na4Pz07,10HzO = 446.1 (13472-36-1) Sodium Taurodeoxycholate Sodium 2-[(3, 12-dihydroxy-
Colourless, slightly effiorescent crystals, freely soluble in 5-cholan-24-oyl)amino]ethanesulfonate monohydrate; 2-
water. [[ (3,5,12)-3,12-Dihydroxy-24-oxocholan-24-
Sodium Rhodizonate Rhodizonic acid disodium salt, yl]amino]ethanesuIfonic acid monosodium salt monohydrate;
[(3,4,5,6-Tetraoxocyc1ohex-I-en-1,2-ylene)dioxy] disodium; C2óH4~aOóS,H20 = 539 .7 (110026-03-4)
CóOóNaz = 214.0 (523-21-7) Content, minimum 94% of CZÓH44NNaOóS,H20.
Violet crystals, soluble in water with an orange-yellow colour. Sodium Tetraborate Borax; disodium tetraborate;
Solutions are unstable and must be prepared on the day of Na zB40 7, 10HzO = 381.4 (1330-43-4)
use. Analytical reagent grade of commerce.
Sodium Salicylate C7H5Na03 = 160.1 (54-21-7) Sodium Tetrahydroborate Sodium borohydride;
Of the British Pharmacopoeia. NaBH4 = 37.8 (16940-66-2)
Sodium Sulfate See Sodium sulfate decahydrate. Colourless, hygroscopic crystals, freely soluble in water,
Sodium Sulfate, Anhydrous Sodium sulphate, soluble in anhydrous ethanol, decomposing at higher
anhydrous; Na ZS04 = 142.0 (7757-82-6) temperature or in the presence of acids or certain metal salts
Ignite at 600 0 to 700 0 anhydrous sodium sulfate complying forming borax and hydrogen.
with the requirements prescribed in the monograph on Store in an airtight container.
Anhydrous sodium sulfate (0099).
Sodium Tetrahydroborate Reducing Solution Stannous Chloride Tin(n) chloride; tin dichloride
Introduce about 100 mL of water into a 500 mL volumetric dehydrate; SnCIz,2H 20 = 225.6 (10025-69-1)
flask containing a stirring bar. Add 5.0 g of sodium hydroxide Content, minimum 97.0% of SnClz,2H 2 0.
in pellets and 2.5 g of sodium tetrahydroborate. Stir until
Colourless crystals, very soluble in water, freely soluble in
complete dissolution, dilute to 500.0 mL with water and mix.
ethanol (96%), in glacial acetic acid and in dilute and
Prepare immediately before use.
concentrated hydrochloric acid.
Sodium Tetraphenylborate (C6Hs)4BNa = 342.2 (143-
Assay Dissolve 0.500 g in 15 mL of hydrochloric acid in a
66-8)
ground-glass-stoppered flask. Add 10 mL of water and 5 mL
White or slightly yellowish, bulky powder, freely soluble in of chloroform. Titrate rapidly with 0.05 M potassium iodate
water and in acetone. until the chloroform layer is colourless.
Sodium T etraphenylborate Solution 1 mL of 0.05 M potassium iodate is equivalent to 22.56 mg of
Filter before use if necessary. SnCI 2,2H 20.
A 10 gIL solution. Stannous Chloride Solution
Use within 1 week. Heat 20 g of tin with 85 mL of hydrochloric acid until no
Sodium Thioglycollate Sodium mercaptoacetate; more hydrogen is released. AlIow to coo!.
C2H3Na02S = 114.1 (367-51-1) Store over an excess of tin, protected from airo
White or almost white, granular powder or crystals, Stannous Chloride Solution Rl
hygroscopic, freely soluble in water and in methanol, slightly Irnmediately before use, dilute 1 volume of stannous chloride
soluble in ethanol (96%). solution with 10 volumes of dilute hydrochloric acid.
Store in an airtight container. Stannous Chloride Solution R2
Sodium Thiosulfate Sodium thiosulphate; To 8 g of stannous chloride add 100 mL of a 20% v/v solution
Na2S203,5H20 = 248.2 (10102-17-7) of hydrochloric acid. Shake until dissolved, heating, if
Of the British Pharmacopoeia. necessary, on a water-bath at 50°. Pass a current of nitrogen
Sodium Thiosulfate, Anhydrous Disodium thiosulfate; for 15 minutes. Prepare immediately before use.
Na2S20 3 = 158 .1 (7772-98-7) Stanolone 17~-Hydroxy-5C(-androstan-3-one;
Content, minimum 98.0%. C19H3002 = 290 .4 (521-18-6)
Sodium Tungstate Disodium tungstate dehydrate; White or almost white powder.
Na2W04,2H20 = 329.9 (10213-10-2) Melting point, about 180°.
White or almost white, crystalline powder or colourless Standard Solution for the Micro Determination of
crystals, freely soluble in water forming a clear solution, Water
practically insoluble in ethanol (96%). Cornmercially available standard solution for the coulometric
Solochrome Dark BIue CI 15705; cal con; mordant black titration of water, containing a certified content of water in a
17; C 2oH)3N2NaOsS = 416.4 (2538-85-4) suitable solvent.
General reagent grade of commerce. Staphylococcus aureus Strain V8 Protease Type XVII-
A brownish black powder with a violet sheen. Produces a B (66676-43-5)
purplish red colour with calcium ions in alkaline solutions. Microbial extracellular proteolytic enzyrne. A Iyophilised
When metal ions are absent, for example, in the presence of powder containing 500 units to 1000 units per mg of solido
an excess of disodium edetate, the solution is blue. Starch
Solochrome Dark Blue Mixture A mixture of 1 part of Potato starch of commerce.
solochrome dark blue with 99 parts of freshly ignited anhydrous Starch, Hydrolysed
sodium sulfate. Electrophoretic grade of commerce.
Complies with the following test. Starch Iodate Paper Irnmerse strips of filter paper in
Sensitivity to calcium Dissolve 0.2 g in 5 mL of water. 100 mL of iodide-free starch solution containing 0.1 g of
To 1 mL of the solution add 50 mL of water, 10 mL of potassium iodate. Drain and allow to dry protected from light.
1M sodium hydroxide and 1 mL of a 1% w/v solution of Starch Iodide Paper Immerse strips of filter paper in
magnesium sulfate; a blue colour is produced. Add 0.1 mL of 100 mL of starch solution containing 0.5 g of potassium iodide.
a 0.15% w/v solution of calcium chloride; a violet colour is Drain and allow to dry protected from light.
produced. Add 0.1 mL of O.OIM disodium edetate VS; apure
Testfor sensitivity Mix 0.05 mL of 0.1 M sodium nitrite
blue colour is produced.
with 4 mL of hydrochloric acid and dilute to 100 mL with
Sorbic Acid Hexa-2,4-dienoic acid; water. Apply one drop of the solution to starch iodide paper;
C 6H s0 2 = 112.1 (110-44-1) a blue spot appears.
General reagent grade of commerce. Starch Mucilage Triturate 0.5 g of starch or soluble starch
Melting point, about 136°. with 5 mL of water and add, stirring continuously, to
Sorbitol D-Sorbitol; C 6H I4 0 6 = 182.2 (50-70-4) sufficient water to produce about 100 mL. Boil for a few
Of the British Pharmacopoeia. minutes, cool and filter.
D-Sorbitol See Sorbitol. Produces a blue colour with free iodine in the presence of a
Squalane 2,6,10,15, 19,23-Hexamethyltetracosane; soluble iodide.
C 30H 6 2 = 422.8 (111-01-3) It must be recently prepared.
Colourless, oily liquid, freely soluble in fatty oils, slightly Starch, Soluble (9005-84-9)
soluble in acetone, in ethanol (96%), in glacial acetic acid White or almost white powder.
and in methano!. Prepare a 20 gIL solution in hot water. The solution is at
d~g, 0.811 to 0.813; n~, 1.451 to 1.453. most slightly opalescent and remains fluid on cooling.
Starch Solution Triturate 1.0 g of soluble starch with 5 mL Melting point, about 170°.
of water and whilst stirring pour the mixture into 100 mL of [alg, about - 51, deterrnined with a 20 giL solution in
boiling water containing 10 mg of mercuric iodide. chloroform.
Carry out the test for sensitivity each time the reagent is Streptomycin Sulfate Streptomycin sulphate;
used. (CzIH39N70d,3HzS04 = 1457 (3810-74-0)
Test/or sensitivity To a mixture of 1 mL of the starch Of the British Pharrnacopoeia.
solution and 20 mL of water, add about 50 mg of potassium Strongly acidic ion-exchange resin See ion-exchange resin,
iodide and 0.05 mL of iodine solution R1. The solution is strongly acidic.
blue.
Strontium Carbonate SrC0 3 = 147.6 (1633-05-2)
Starch Solution, Iodide-free Prepare the solution as
prescribed for starch solution omitting the mercuric iodide. White or almost white, ctystalline powder.
Prepare immediately before use. Content, minimum99.5% .
Starch Solution Rl Mix 1 g of soluble starch and a small Strontium Chloride SrCl z,6H zO = 266 .6 (10025-70-4)
amount of cold water. Add this mixture, while stirring, to Analytical reagent grade of commerce.
200 mL of boiling water. Add 0.25 g of salicylic acid and boil Strontium Chloride Hexahydrate
for 3 minutes. Immediately remove from the heat and cool. SrCl z,6H zO = 266.6 (10025-70-4)
If long storage is required, the solution shall be stored at 4° White or almost white ctystals, very soluble in water.
to 10°. A fresh starch solution shall be prepared when the Melting point, about 115° (loss ofwater) and 872°.
end-point of the titration from blue to colourless fails to be
sharp. If stored under refrigeration, the starch solution is Strontium Selective Extraction Resin
stable for about 2 to 3 weeks. Commercially available resin prepared by loading a
Test/or sensitivity A mixture of 2mL of starch suspension of 4,4 ' (5')-di-tert-butylcyclohexano-18-crown-6
solution R1, 20 mL of water, about 50 mg of potassium iodide (crown ether) in octanol onto an inert chromatographic
and 0.05 mL of iodine solution R1 is blue. support. The bed density of this resin is approximately
0.35 glmL.
Starch Solution R2 Triturate 1.0 g of soluble starch with
5 mL of water and whilst stirring pour the mixture into Strontium-85 Spiking Solution
100 mL of boiling water. U se a freshly prepared solution. Dilute strontium-85 standard solution to a radioactivity
Test/or sensitivity To a mixture of 1 mL of the starch concentration of approximately 10 kBq/mL with a 0.27 gIL
solution and 20 mL of water, add about 50 mg of potassium solution of strontium chloride hexahydrate in a 1.03 giL
iodide and 0.05 mL of iodine solution R1. The solution is solution of hydrochloric acid.
blue. Strontium-85 Standard Solution
Starch Substrate Determine the water content of A solution of strontium-85 in the form of Srz+ ions in a
starch EPBRP by heating at 120° for 4 hours. Stir a quantity 51.5 gIL solution of hydrochloric acid.
of starch EPBRP equivalent to 2.0 g of the dried substance Styrene Ethenylbenzene; CsHs = 104.2 (100-42-5)
with 10 mL of water and add, stirring continuously, to Boiling point, about 145°.
160 mL of boiling water. Rinse the container with several 10-
Colourless, oily liquid, very slightly soluble in water.
mL quantities of water, add the washings to the hot starch
solution and heat to boiling, stirring continuously. Cool to Styrene-Divinylbenzene Copolymer
20° and add sufficient water to produce 200 mL. Porous, rigid, cross-linked polymer beads. Several grades are
Use on the day of preparation. available with different sizes of beads. The size range of the
beads is specified after the name of the reagent in the tests
Stearic Acid Octadecanoic acid; ClsH360Z = 284.5 (57-
where it is used.
11-4)
Succinic Acid Butanedioic acid; C 4 H 60 4 = 118.1 (110-
White or almost white powder or flakes, greasy to the touch,
15-6)
practically insoluble in water, soluble in hot ethanol (96%).
White or almost white, crystalline powder or colourless
crystals, soluble in water and in ethanol (96%) .
Stearic acid used in the assay of total fatty acids in Saw palmetto
fruit (1848) complies with the following additional test.
Sucrose C1zHzzOll = 342.3 (57-50-1)
monograph Saw palmetto fruit (1848) . Of the British Pharrnacopoeia
Content, minimum 98%, calculated by the norrnalisation Sudan Orange CI 12055, 1-(phenylazo)naphthalen-2-01,
procedure. sudan I, sudan yellow; Cl6HI ZNzO = 248.3 (84i-07-9)
Stearic Anhydride C36H7003 = 551.0 (638-08-4) Orange-red powder, practically insoluble in water, soluble in
methylene chloride.
White, waxy flakes; melting point, about 70°.
Sudan Red CI 26100; sudan III; solvent red 23; 1-
Stearyl Alcohol 1-0ctadecanol;
(4-phenylazophenylazo )-2-naphthol;
C 1sH 3SO = 270 .5 (112-92-5)
CZZHl6N40 = 352.4 (85-86-9)
Technical reagent grade of commerce.
A reddish brown powder.
Stigmasterol (22E)-Stigmasta-5,22-dien-3~-01; (22E)-24-
Sudan Red G CI 12150; sudan red I; 1-(2'-
ethylcholesta-5,22-dien-3~-01; C z9 H 4S O = 412.7 (83-48-7)
methoxyphenylazo)-2-naphthol; C17HI4NzOz = 278 .3
White or almost white powder, insoluble in water.
Reddish-brown powder, practically insoluble in water.
Chromatography Thin-layer chromatography (2.2.27) Sulfomolybdic Reagent R3 Sulphomolybdic reagent R3 .

using silica gel G as the coating substance: apply 10 ¡¡L of a Dissolve with heating 2.5 g of ammonium molybdate in 20 mL
0.1 gIL solution in methylene chloride and develop over a path of water. Dilute 28 mL of sulfuric acid in 50 mL of water,
of 10 cm with the same solvent; the chromatogram shows then cool. Mix the two solutions and dilute to 100 mL with
only one principal spot. water.
Sudan Red Solution A 0.5% w/v solution of sudan red in Store in a polyethylene container.
anhydrous acetic acid.
Sulfosalicylic Acid 2-Hydroxy-5-sulfobenzoic acid; 5-
Sudan YelIow See sudan orange. Sulfo-2-hydroxybenzoic acid; 5-sulpho-2-hydroxybenzoic
Sudan YelIow Solution A 0.2% w/v solution of sudan acid, 3-sulpho-6-hydroxybenzoic acid; 3-sulpho-6-
yellow in a mixture of 1 volume of benzene and 4 volumes of hydroxybenzoic acid; sulfosalicylic acid;
petroleum spirit (boz1ing range, 60" to 80"). C 7H 6 0 6S,2H 20 = 254 .2 (5965-83-3)
Sulfanilamide 4-Aminobenzenesulfonamide, 4- White or almost white, crystalline powder or crystals, very
aminobenzenesulphonamide, sulphanilamide; soluble in water and in ethanol (96%).
C 6 H aN 2 0 2 S = 172.2 (63-74-1) Melting point, about 109°.
White or almost white powder, slightly soluble in water, Sulfur Sulfur for External Use of the British
freely soluble in boiling water, in acetone, in dilute acids and Pharmacopoeia.
in solutions of the alkali hydroxides, sparingly soluble in
Sulfur Dioxide Sulphur dioxide, sulfurous anhydride;
ethanol (96%) and in light petroleum.
S02 = 64.05 (7446-09-5)
A colourless gas. When compressed it is a colourless liquido
Sulfathiazole 4-Amino-N-(thiazol-2-
Sulfur Dioxide Rl Sulphur dioxide R1 ;
yl)benzenesulfonamide; C9H9N302S2 = 255 .3 (72-14-0) S02 = 64.1 (7446-09-5)
White or yellowish-white powder or crystals, very slightly
Content, minimum 99 .9% v/v.
soluble in water, soluble in acetone, slightly soluble in
ethanol (96%). It dissolves in dilute mineral acids and in Sulfur Dioxide Solution Sulphur dioxide solution,
solutions of alkali hydroxides and carbonates. sulfurous acid, sulphurous acid; H 2S0 3 = 82.08
Melting point, about 200°. Use a solution of commerce containing about 5% w/v of
S02; weight per mL, about 1.03 g.
Sulfamic Acid Sulphamic acid;
H 3N0 3 S = 97.1 (5329-14-6) Sulfuric Acid Sulphuric acid; H 2S0 4 = 98.08 (7664-93-9)
White or almost white crystalline powder or crystals, freely Content, 95.0% mlm to 97.0% mlm.
soluble in water, sparingly soluble in acetone, in ethanol Colourless, caustic liquid with an oily consistency, highly
(96%) and in methanol. hygroscopic, miscible with water and with ethanol (96%)
Melting point, about 205°, with decomposition. producing intense heat.
d~g, 1.834 to 1.837.
Sulfan Blue CI 42045; patent blue VF; acid blue 1;
disulfine blue; sodium [[[(4-diethylamino)phenyl] A 10 gIL solution is strongly acid and gives the reactions of
(2,4isulfonatophenyl)methylene] cyclohexa-2,5-dien-1- sulfates (2.3.1).
ylidene]diethylammonium, sulphan blue; Appearance Ir is clear (2.2. 1) and colourless (2.2.2,
C27H31N2Na06S2 = 566.6 (129-17-9) Method JJ).
Violet powder, soluble in water. Dilute solutions are blue and Oxidisable substances Pour 20 g cautiously, with
turn yellow on the addition of concentrated hydrochloric cooling, into 40 mL of water. Add 0.5 mL of 0.002 M
acid. potassium permanganate. The violet colour persists for at least
Sulfanilic Acid 4-Aminobenzenesulfonic acid; sulphanilic 5 minutes.
acid; C 6 H 7N0 3S = 173.2 (121-57-3) Chlorides Maximum 0.5 ppm.
Colourless crystals, sparingly soluble in water, practically Pour 10 g, carefully and while cooling, into 10 mL of water
insoluble in ethanol (96%). and after cooling dilute to 20 mL with the same solvento
Sulfanilic Acid Solution Sulphanilic acid solution. Add 0.5 mL of silver nitrate solutian R2. Allow to stand for
Dissolve 0.33 g of sulfanilic acid in 75 mL of water heating 2 minutes protected from bright light. The solution is not
gently if necessary and dilute to 100 mL with glacial acetic more opalescent than a standard prepared at the same time
acid. ' using a mixture of 1 mL of chlonde standard solution (5 ppm
Sulfanilic Acid Solution Rl Sulphanilic acid solution Rl. CV, 19 mL of water and 0.5 mL of silver nitrate solution R2.
Dissolve 0.5 g of sulfanilic acid in a mixture of 75 mL of Nitrates Maximum 0.5 ppm.
dz1ute acetic acid and 75 mL of water. Pour 50 g or 27.2 mL, carefully and while cooling, into
Sulfanilic Acid Solution, Diazotised Sulphanilic acid 15 mL of water. Add 0.2 mL of a freshly prepared 50 giL
solution, diazotised. solution of brucine in glacial acetic acid. After 5 min any
Dissolve, with warming, 0.9 g of sulfanilic acid in 9 mL of colour is less intense than that of a reference mixture
hydrochloric acid, and dilute to 100 mL with water. Cool prepared in the same manner and containing 12.5 mL of
10 mL of this solution in iced water and add 10 mL of an water, 50 g of nitrogen-free sulfuric acid, 2.5 mL of nitrate
ice-cold 45 gIL solution of sodium nitrite. Allow to stand at 0° standard solution (JO ppm NO~ and 0.2 mL of a 50 gIL
for 15 min (if stored at this temperature, the solution is solution of brucine in glacial acetic acid.
stable for 3 days) and immediately before use add 20 mL of Ammonium Maximum 2 ppm.
a 100 gIL solution of sodium carbonate. Pour 2.5 g, carefully and while cooling, into water and dilute
Sulfomolybdic Reagent R2 Sulphomolybdic reagent R2. to 20 mL with the same solvento Cool, and add dropwise
Dissolve about 50 mg of ammonium molybdate in 10 mL of 10 mL of a 200 gIL solution of sodium hydroxide, followed by
sulfuric acid.
1 mL of alkaline potassiu111 tetraiodomercurate soluciono Titrate with 1 M sodium hydroxide, using 0.1 mL of methyl
The colour of the solution is !ess intense than that of a red solution as indicator.
mixture of 5 mL of ammonium standard solution (1 PP111 1 mL of 1 M sodium hydroxide is equivalent to 49.04 mg of
NH,J, 15 mL of water, 10 mL of a 200 gIL solution of H 2 S0 4 ·
sodiu111 hydroxide and 1 mL of alkaline potassium Sulfuric Acid, Ethanolic Sulphuric acid, ethanolic
tetraiodomercurate solution.
Solutions of the requisite molarity may be obtained by
Arsenic (2.4.2, Method A) Maximum 0.02 ppm. mixing sulfun·c acid with ethanol (96%) as directed under
To 50 g add 3 mL of nitric acid and evaporate carefully until sulfuric acid.
the volume is reduced to about 10 mL. Cool, add to the When 'ethanolic sulfun·c acid' is followed by a percentage
residue 20 mL of water and concentrate to 5 mL. Prepare the figure, an instruction to use sulfun·c acid diluted with ethanol
standard using 1.0 mL of arsenic standard solution (1 ppm As). (96%) to produce the specified percentage v/v proportion of
¡ron (2.4.9) Maximum 1 ppm. sulfuric acid is implied.
Dissolve the residue on ignition with slight heating in 1 mL Sulfuric Acid-Formaldehyde Reagent Sulphuric acid-
of dilute hydrochloric acid and dilute to 50.0 mL with water. formaldehyde reagent
Dilute 5 mL of this solution to 10 mL with water. Mix 2 mL of formaldehyde solution with 100 mL of sulfuric
Heavy metals (2.4.8) Maximum 2 ppm. acid.
Dilute 10 mL of the solution obtained in the test for iron to Sulfuric Acid, Heavy Metal-free Sulphuric acid, heavy
20 mL with water. 12 mL of the solution complies with test metal-free
A. Prepare the reference solution using lead standard solution Complies with the requirements prescribed for sulfuric acid
(2 ppm Pb) . with the following maximum contents of heavy metals:
Residue on ignition Maximum 0.001 %, determined on As: 0.005 ppm; Cd: 0.002 ppm; Cu: 0.001 ppm; Fe:
100 g by evaporating cautiously in a small crucible over a 0.05 ppm; Hg: 0.005 ppm; Ni: 0.002 ppm; Pb: 0.001 ppm;
naked flame and igniting the residue to redness. Zn: 0.005 ppm.
Assay Weigh accurately a ground-glass-stoppered flask Sulfuric Acid, Methanolic Sulphuric acid, methanolic
containing 30 mL of water, introduce 0.8 mL of the sulfuric
Solutions of the requisite molarity may be obtained by
acid, cool and weigh again. Titrate with 1 M sodium
mixing sulfuric acid with methanol as directed under sulfuric
hydroxide, using 0.1 mL of methyl red solutwn as indicator.
acid.
1 mL of 1 M sodium hydroxide is equivalent to 49.04 mg of
When '111ethanolic sulfuric acid' is followed by a percentage
H ZS04.
figure, an instruction to use sulfuric acid diluted with methanol
Store in a ground-glass-stoppered container made of glass or to produce the specified percentage v/v proportion of sulfuric
other inert material. acid is implied.
When no molarity is indicated use analytical reagent grade of Sulfuric Acid, Nitrogen-free
commerce containing about 96% w/w of sulfuric acid and
Sulphuric acid, nitro gen-free
about IBM in strength.
Complies with the requirements prescribed for sulfuric acid
When solutions of molarity XM are required, they should be
with the following additional test.
prepared by carefully adding 54x mL of sulfuric acid to an
equal volume of water and diluting to 1000 mL with water. Nitrates To 5 mL of water add carefully 45 mL of the
sulfuric acid, allow to cool ro 40° and add 8 mg of
When 'suifuric acid' is followed by a percentage figure, an
diphenylbenzidine. The solution is faint pink or very pale blue.
instruction to add, carefully, sulfuric acid to water to produce
the specified percentage v/v (or, if required, w/w) proportion Sulfuric acid, nitrogen-free Rl
of sulfuric acid is implied. Complies with the requirements prescribed for nitrogen-free
Sulfuric Acid, 2.5M Alcoholic Sulphuric acid, 2.5M sulfuric acid.
alcoholic Content, 95.0% mlm to 95.5% m/m.
Carefully and with constant cooling, stir 14 mL of sulfuric Sunflower Oil Refined Sunflower Oil of the British
acid into 60 mL of anhydrous ethanol. Allow to cool and Pharmacopoeia.
dilute to 100 mL with anhydrous ethanol. Prepare immediately Swertiamarin (4R,5R,6S)-5-Ethenyl-6-(p-n-
before use. glucopyranosyloxy )-4a-hydroxy-4,4a, 5, 6-tetrahydro-1 H,3H-
Sulfuric Acid, O.25M Alcoholic Sulphuric acid, 0.25M pyrano [3,4-c1pyran-1-one, swertiamaroside;
alcoholic C1 6H2Z01O = 374.3 (17388-39-5)
Dilute 10 mL of 2.5 M alcoholic sulfuric acid to 100 mL with Re Tagatose n-(yxo-Hexulose;
anhydrous ethanol. Prepare immediately before use. C 6H 12 0 6 = 180.16 (87-81-0)
Sulfuric Acid, Alcoholic Solution of Sulphuric acid, General reagent grade of commerce.
alcoholic solution of Melting point, 134° to 135°; [al~o, - 2.3 (2.19% w/v solution
Carefully and with constant cooling, stir 20 mL of sulfuric in water).
acid into 60 mL of ethanol (96%). Allow to cool and dilute to Talc (14807-96-6)
100 mL with ethanol (96%). Prepare immediately before use. Purified grade of commerce.
Sulfuric Acid, Dilute Sulphuric acid, dilute; A very fine, white powder.
Contains 98 giL of H 2S0 4 . Tannic Acid (1401-55-4)
Add 5.5 mL of sulfuric acid to 60 mL of water, allow to cool General reagent grade of commerce.
and dilute to 100 mL with the same solvent. Yellowish to light brown, glistening scales or amorphous
Assay lnto a ground-glass-stoppered flask containing powder.
30 mL of water, introduce 10.0 mL of the di/ute sulfun·c acid. Store protected from light.
Tannic Acid Reagent Dissolve 25 mg of tannic acid in (X - Terpineol L/sed in gas chromatography complies with the
20 mL of glacial acetic acid and add 80 mL of orthophosphoric following test.

acid. Assay Examine by gas chl'Omatography, Appendix III B,
Prepare immediately before use. under the conditions described in the test for
Tanshinone HA 1,6,6-Trimethyl-6,7,8,9- Chromatographic profile in the monograph for Anise Oil
tetrahydrophenanthro [1 ,2-b]furan-l O, ll-dione; using as solution (1) a 10% w/v solution of the reagent being
Cl9Hl S0 3 = 294.3 (568-72-9) examined in hexane.
Tartaric Acid See (+)-Tartaric acid. The area of the principal peak is not les s than 97.0% of the
total area of the peaks. Disregard the peak due to hexane.
(+ )- Tartaric Acid C4H 60 6 = 150.1 (87-69-4)
Terpinolene p-Mentha-l,4(8)-diene; 4-isopropylidene-l-
methylcyclohexene; ClOHl6 = 136.2 (586-62-9)
Taurodeoxycholic Acid Sodium Salt
General reagent grade of cornmerce.
CZ6H44NaN06S = 521.7 (1180-95-6)
d¡O, about 0.863; n~, about 1.488; boiling point, about
184°.
Taxifolin (2R,3R)-2-(3,4-Dihydroxyphenyl)-3,5, 7-
Terpinolene used in gas chromatography complies with the
trihydroxy-2,3-dihydro-4H-l-benzopyran-4-one;
following test.
ClsHIZ07 = 304.3 (480-18-2)
Assay Carry out the method for Chromatographic profile
White or almost white powder, slightly soluble in ethano!.
described in the monograph for Tea Tree Oi!. The content
A solution in ethanol shows an absorption maximum (2.2.25) is not less than 90%, calculated by the normalisation
at 290 nm. procedure.
Tecnazene C 6HCI4N0 2 = 260.9 (117-18-0) Testosterone Cl9H2S0Z = 288.4 (58-22-0)
Use a grade of commerce suitable for pesticide residue General reagent grade of commerce.
analysis. A suitable certified reference solution (lO ng/~IL in
cyclohexane) may be used.
Testosterone Propionate C 22 H 32 0 3 = 344.5 (57-85-2)
Terpinene l-Isopropyl-4-methylcyclohexa-l,3-diene;
CIOHI6= 136.2 (99-86-5) General reagent grade of commerce.
0
General reagent of commerce. Melting point, about 121 •
d¡O, about 0.837; n~o, about 1.478; boiling point, about 1,2,3 ,4-Tetra-O-acetyl-~-D-glucopyranose
174°. C14HzoOlO = 348.3 (13100-46-4)
IJ.- Terpinene L/sed in gas chromatography complies with the
White or almost white powder, soluble in water with gentle
following test. heating.
Assay Carry out the method for Chromatographic profile [a]~, + 11 , determined on a 6 gIL solution in chloroform;
described in the monograph for Tea Tree Oil. The content melting point, 126 0 to 128°.
is not less than 95%, calculated by the normalisation 1,3 ,4,6- Tetra-O-acetyl-~-D-mannopyranose
procedure. CI4HzoOIO'= 348.3 (18968-05-3).
y- Terpinene l-Isopropyl-4-methylcyclohexa- l,4-diene; Colourless or white powder or crystals.
ClOHl6 = 136.2 (99-85-4) Melting point, 160° to 161°.
dr,
186°.
about 0.850; ni], 1.474 to 1.475; boiling point, 183 to
0
[al~o, - 68, determined on a 7 gIL solution in methylene
chloride.
Tetrabutylammonium Bromide
y- Terpinene L/sed in gas chromatography complies with C 16H 36 BrN = 322.4 (1643-19-2)
thefollowing test.
Assay Examine by gas chromatography as described in the
monograph for Peppermint Oil using the reagent being Melting point, 102° to 104°.
examined as the test solution. The area of the principal peak Tetrabutylammonium Dihydrogen Orthophosphate
is not less than 90.0% by nonnalisation. Tetrabutylammonium dihydrogen phosphate;
Terpinen-4-o1 4-Methyl-l-( l-methylethyl)cyclohex-3,- en-l- Cl6H3SN04P = 339.5 (5574-97-0)
01; ClOH1SO = 154,2 (562-74-3) General reagent grade of commerce.
General reagent grade of commerce. Melting point, about 153°.
d~g, about 0.934; n~o, about 1.477; boiling point, 209° to Store in an airtight container.
212°, Tetrabutylammonium Dihydrogen Phosphate See
Terpinen-4-ol L/sed in gas chromatography complies with the TetrabutylammoniL/m dihydrogen orthophosphate.
following test. Tetrabutylammonium Hydrogen Sulfate
Assay Examine by gas chromatography as described in the Tetrabutylammonium hydrogen sulphate;
monograph for Lavender Oil using the reagent being Cl6H37N04S = 339.5 (32503-27-8)
examined as the test solution. The area of the principal peak Analytical reagent grade of commerce.
is not less than 98.0% by normalisation. A white, crystalline powder; melting point, 169° to 173°.
Ci.- Terpineol (RS)-2-( 4-Methylcyclohex-3-enyl)propan-2-01; Complies with the following tests.
ClOH1SO = 154.2 (98-55-5) Absorbance Absorbance of a 5.0 % w/v solution in the
General reagent grade of commerce. range 240 nm to 300 nm, Appendix II B, not greater than
It may contain 1% to 3% of ~-terpineo!. 0.05.
Melting point, about 35°; dlg, about 0.935; n~o, about 1.483; Acidity A 1.7% w/v solution has a pH of about 1.5,
[al~o, about 92.5. Appendix V L.
Tetrabutylarnmonium Hydrogen Sulfate R1 n- Tetradecane C l4 H 30 = 198.4 (629-59-4)

Tetrabutylammonium hydrogen sulphate R1 General reagent grade of commerce containing not les s than
Complies with the requirements prescribed for 99.5% of C 14H 30.
tetrabutylammonium hydrogen sulfate and with the following A colourless liquid; dig, about 0.76; n~, about 1.429;
additional requirement: melting point, about _5°; boiling point, about 253°.
Absorbance The absorbance of a 50 gIL solution, at Tetradecylarnmonium Bromide
wavelengths from 215 nm to 300 nm, is not greater than C 4oH s4BrN = 659.0 (14937-42-9)
0.02. Chromatographic reagent grade of commerce.
Tetrabutylammonium Hydroxide Melting point, 88° to 89°.
C1 6H37NO,30H20 = 800 (2052-49-5)
Tetraethylammonium Hydrogen Sulfate
General reagent grade of commerce containing not less than Tetraethylammonium hydrogen sulphate;
98.0% w/v of CI6H37NO,30H20. C sH 21 N0 4S = 227.3 (16873-13-5)
Assay Dissolve 1 g in 100 mL of water and titrate
immediately with O.lM hydrochloric acid VS determining the
end point potentiometrically. Each mL of O.IM hydrochloric
acid VS is equivalent to 80.0 mg of CI6H37NO,30H20 . Tetraethylammonium Hydroxide Solution
Tetrabuty1ammonium Hydroxide Solution C SH 21 NO = 147.3 (77-98-5)
General reagent grade of commerce containing 40.0% w/v of Chromatographic reagent grade of commerce containing
20% w/v of C SH 21 NO.
C 16H 37NO.
Tetrabuty1arnmonium Hydroxide Solution (104 gIL) dig, about 1.01 ; n5°,about 1.372.
Dilute tetrabutylammonium hydroxide to contain 10.4% w/v of Tetraethylene Pentamine 3,6,9-Triazaundecan-l ,1l-
C 16H 37NO . diamine; C SH 21N s = 189.3 (112-57-2)
Tetrabutylammonium Hydroxide Solution (400 gIL) General reagent grade of commerce.
See Tetrabutylammonium hydroxide solution n~, about 1.506.
Tetrabutylammonium Hydroxide, O.4M Store protected from humidity and heat.
Use a grade of commerce suitable for chromatography. Tetraheptylammonium Bromide
Tetrabutylammonium Iodide Cl 6H3óIN = 369.4 (311- C 2sH 60 BrN = 490 .7 (4368-51-8)
28-4) Chromatographic reagent grade of commerce.
General reagent grade of commerce containing not less than Melting point, 89° to 91 °.
98.0% of C 1óH 36IN. Tetrahexylammonium Hydrogen Sulfate
Complies with the following tests. Tetrahexylammonium hydrogen sulphate;
Sulfated ash Not more than 0.02%. C24Hs2N,HS04 = 451.8 (32503-34-7)
Assay Dissolve 1.2 g in 30 mL of water and add 50 mL of Chromatographic reagent grade of cornmerce.
O.IM silver nitrate VS and 5 mL of 2M nitric acid. Titrate the Melting point, 100° to 102°.
excess of silver nitrate with O.IM ammonium thiocyanate VS Tetrahydrofuran Tetramethylene oxide;
using 2 mL of ammonium iron(m) sulfate solution R2 as C4 H sO = 72.11 (109-99-9)
indicator. Each mL of 0.1M silver nitrate VS is equivalent to Analytical reagent grade of commerce.
36.94 mg of C 16H 3óIN.
A clear, colourless, flammable liquid; boiling point, about
Tetrachloroethane 1,1,2,2-Tetrachloroethane;
C 2H 2CI 4 = 167.9 (79-34-5)
66°; d~g, about 0.89 .
Do not distil unless it complies with the following test.
Peroxides Place 8 mL of potassium iodide and starch
dig, about 1.59; n~ , about 1.495. Not less than 95% distils solution in a ground-glass-stoppered cylinder with a capacity
between 145° and 147°.
of 12 mL and about 1.5 cm in diameter and add sufficient
Tetrachlorvinphos C lO H 9 CI 40 4P = 366.0 (22248-79-9) of the substance being examined to fill the cylinder
Use a grade of cornmerce suitable for pesticide residue completely, shake vigorously and allow to stand for
analysis. A suitable certified reference solution (10 ng/¡.tL in 30 minutes protected from light. No colour is produced.
iso-octane) may be used. Tetrahydrofuran used in spectrophotometry complies with the
Melting point, about 95 °. following test.
Tetracos-15-enoic Acid Methyl Ester 15-Tetracosaenoic Transmittance Not less than 20% at 255 nm, 80% at
acid methyl ester; Methyl tetracos-15- enoate; Nervonic acid 270 nm and 98% at 310 nm determined using water in the
methyl ester; C2sH4S02 = 380.7 (2733-88-2) reference cel!.
Contains not less than 99.0% of C2sH4S0 2, determined by Tetrahydrofuran for Chromatography It complies with
gas chromatography. the requirements of tetrahydrofuran and with the following
Liquid. requirements:
Tetracycline C22H24N20 S = 444.4 (60-54-8) diO, 0.8892; boiling point, about 66°.
General reagent grade of commerce. Contains not less than 99 .8% of C 4H sO.
Melting point, about 176°. Tetrahydrofuran, Stabiliser-free Tetrahydrofuran that is
Store protected from light. free from stabilisers and inhibitors.
Tetracycline Hydrochloride Of the British Reagent grade of commerce.
Pharmacopoeia.
D- Tetrahydropahnatine Hydrochloride Tetramethylsilane (CH3)4Si = 88.23 (75-76-3)

C 21 H zsN0 4 ,HCI = 391.90 (6024-83-5) Spectroscopic reagent grade of commerce.
General reagent grade of commerce. A colourless liquid; dig, about 0.64; ni?, about 1.358; boiling
(J.- Tetralone 1-0xotetraline; 3,4-Dihydronaphthalen- point, about 26°.
1(2H)-one; CIOHIOO = 146.2 (529-34-0) When used in nuclear magnetic spectrometry, complies with the
Boiling point, about 115°C; melting point, about 5°C. following test.
Tetramethylammonium Chloride Nuclear magnetic resonance In the NMR spectrum of a
C 4 H 1Z CIN = 109.6 (75-57-0) 10% v/v solution of the reagent being examined in
General reagent grade of commerce. deuterochloroform, the intensity of any foreign signal, excluding
Melting point, about 300°, with decomposition. those due to spi=ing side bands and to chloroform, is not
greater than the intensity of the I3C-satellite signals located
Tetramethylammonium Hydrogen Sulfate at a distance of 59.1 Hz on each side of the principal signal
Tetramethylarnmonium hydrogen sulphate; of tetramethylsilane.
C 4 H 13 N0 4S = 171.2 (80526-82-5)
Tetrandrine C3sH41N106 = 623 (518-34-3)
Chromatographic grade of commerce.
1,2,3,4-Tetraphenylcyclopenta-l ,3-diene
Melting point, about 295°. C 29 H zz = 370.5 (15570-45-3)
Tetramethylammonium hydrogen sulfate used in liquid
chromatography complies with the following test.
Transmittance Not less than 50% at 200 nm and 90% at
220 nm determined using a 0.005M solution. 1,2,3,4-Tetraphenylcyclopenta-l ,3-dienone
Tetraphenylcyclopentadienone; C z9 H 20 0 = 384.5 (479-33-4)
Tetramethylammonium Hydroxide See
Tetramethylammonium hydroxide pentahydrate.
TetramethyIammonium Hydroxide Pentahydrate Melting point, about 218°.
C4 H 13 NO,5H zO = 181.2 (10424-65-4) Tetraphenylethylene C16H zo = 332.5 (632-51-9)
TetramethyIammonium Hydroxide Solution Tetrapropylammonium Chloride
General reagent grade of commerce containing not less than C11HzsCIN = 221.8 (5810-42-4)
0
10.0% w/w of C 4H 13 NO. White, crystalline powder; melting point, about 241 •
A c1ear, colourless or very pale Iiquid. Tetrazolium Blue Blue tetrazolium salt; 3,3'-(3,3 '-
dimethoxy-4,4'-biphenylylene )bis(2,5-diphenyl- 2H-
Assay To 1 g add 50 mL of water and titrate with
0.05M sulfuric acid VS using methyl red solutwn as indicator. tetrazolium chloride); C4oH32ClzNsOz = 727.7 (1871- 22-3)
Each mL of 0.05M sulfuric acid VS ís equivalent to 9.115 mg General reagent grade of commerce.
of C4 H 13 NO. Yellow crystals; melting point, about 245°, with
Tetramethylammonium Hydroxide Solution, Dilute decomposition.
Dilute 10 mL of tetramethylammonium hydroxide solution to Tetrazolium Blue Solution, AIkaline Immediately before
100 mL with aldehyde-free ethanol (96%) . It contains about use mix l volume of a 0.2% w/v solution of tetrazolium blue
1% w/v of C 4 H 13 NO. in methanol with 3 volumes of a 12% w/v solution of sodium
Prepare irnmediately before use . hydroxide in methanol.
Tetramethylbenzidine 3,3',5,5'-Tetramethylbiphenyl- Tetrazolium Bromide 3-(4,5-Dimethylthiazol-2-yl)-2,5-
4,4'-diamine; C16HzoNz = 240.3 (54827-17-7) diphenyltetrazolium bromide; MTT;
General reagent grade of commerce. ClsH16BrNsS = 414.3 (298-93-1)
•
Tetrazolium salt 5-(3-Carboxyrnethoxyphenyl)-3-(4,5-
1,1,3,3-Tetramethylbutylamine 2-Amino-2,4,4- dimethylthiazol-2-yl)-2-( 4-sulfophenyl)- 2H- tetrazolium, i=er
trimethylpentane; C SH 19 N = 129.3 (107-45-9) salt, MTS; CzoH17Ns06S2 = 487.5 (138169-43-4)
General reagent grade of commerce. Thallium(r) Nitrate Thallous nitrate;
dig, about 0.805; ntO, about 1.424; boiling point, about TIN0 3 = 266.4 (10102-45-1)
140°. General reagent grade of commerce.
T etramethyldiaminodiphenyhnethane See 4,4'- Thallium(r) Sulfate Thallium(I) sulphate, thallous sulfate,
Methylenebis-N,N-dimethylaniline. thallous sulphate; TlzS0 4 = 504.8 (7446-18-6)
Tetramethyldiaminodiphenyhnethane Reagent See White, rhomboid prisms.
4,4'-Methylenebis-N,N-dimethylaniline reagent. Thallous Sulfate See Thallium(I) sulfate.
T etramethylethylenediamine Tetramethylethane-l,2- Thebaine (5R,9R,13S)-4,5-Epoxy-3,6-dimethoxy-9a-
diamine; N,N,N' ,N'-tetramethylethylenediamine; methylmorphina-6,8-diene; (C 19 H z1 N)3 = 311.4 (115-37-7)
C6H16NZ = 116 .2 (110-18-9) General reagent grade of commerce.
General reagent grade of commerce. A white or pale yellow powder; melting point, about 193 o .
A colourless liquid; boiling point, about 121 °; dig, about Complies with the following test.
0.78. Chromatography Carry out Identification test B in the
N,N,N' ,N'- Tetramethyl-p-phenylenediamine monograph for Opium applying to the plate as a band
Dihydrochloride C IO H 16N z,2HCI = 237.2 (637-01-4) (20 mm x 3 mm) 20 ¡.tL of a 0.05% w/v solution. The
General reagent grade of commerce. chromatogram obtained shows an orange-red or red principal
Whitish grey crystals . spot with a Rf value of about 0.5 .
Theobromine Of the British Pharmaeopoeia. Thrombin Human thrombin

Theophylline C 7 H sN 4 0 2 = 180.2 (58-55-9) Dried human thrombin obtained from liquid plasma. It may
General reagent grade of eommeree. be prepared by precipitation with suitable salts and organic
solvents under controlled conditions of pH, ionie strength
and temperature.
Thiamazo1e Methimazole; l_Methyl-1H-imidazole-2-thiol;
C 4H 6N 2S = 114.2 (60-56-0)
A yellowish white powder.
General reagent grade of commeree.
Store in a sealed, sterile container under nitrogen, protected
A white or almost white, crystalline powder; melting point,
from light and at a temperature below 25°.
about 145°.
Thrombin, Bovine A preparation of the enzyrne that
2-(2- Thienyl)acetic Acid 2-Thiopheneacetic acid;
C 6H 60 2S = 142.1 (1918-77-0) converts fibrinogen into fibrin.
Melting point, about 650
•
Thrombin, Human See Thrombin.
Thrombin Solution Human thrombin solution
Thioacetamide C 2H 5NS = 75.13 (62-55-5)
General reagent grade of commerce. Reconstitute thrombin as direeted by the manufacturer and
dilute with tris-chloride buffer pH 7.4 to contain 5 IU per rnL.
White crystaIs or crystalline powder; melting point, about
Store below O°.
113°.
Thioacetamide Reagent Add 1 mL of a mixture of Thrombin Solution, Human Rl
15 mL of 1M sodium hydroxide, 5 mL of water and 20 rnL of Reconstitute human thrombin as directed by the manufacturer
glycerol (85%) to 0.2 mL of thioacetamide solution, heat in a and dilute to 2.5 IU/roL with phosphate buffer solution pH 6.5.
water bath for 20 seconds, cool and use immediately. Thrombin Solution, Human See Thrombin solution.
Thioacetamide Solution A 4% w/v solution of Thromboplastin
thioacetamide. A preparation containing the membrane glycoprotein tissue
Thiobarbituric Acid 4,6-Dihydroxy-2- factor and phospholipid, either purified from animal brain
mercaptopyrimidine; C 4H 4 N 20 2S = 144.2 (504-17-6) (usually rabbit) or human placenta or manufactured using
General reagent grade of commerce. recombinant DNA technology with added phospholipid. The
Thiodiethylene Glycol See Thiodiglycol. preparation is formulated for routine use in the prothrombin
time test and may contain calcium.
Thiodiglycol Thiodiethylene glyeol; 2,2'-thiodiethanol;
C 4H IO 0 2S = 122.2 (111-48-8) Thromboplastin Reagent Thrombokinase extract
General reagent grade of commerce. Extract 1.5 g of acetone-dried ox brain with 60 mL of water
for 10 to 15 minutes at 50°, centifuge for 2 minutes at
n~, about l.522 .
1500 revolutions per minute and decant the supematant
Thioglycollic Acid See Mercaptoacetic acid. liquid. This extract will retain its activity for several days
Thiomalic acid (2RS)-2-Sulfanylbutanedioie acid; when stored in a refrigerator. It may contain 0.3% w/v of 0-
C 4H 60 4S = 150.2 (70-49-5) cresol as an antirnierobial preservative.
Melting point, 150° to 152°. Thujone 4-Methyl-l-(I-methylethyl)bicyclo[3.1.0]hexan-3-
Thiomersal CgHgHgNa02S = 404.8 (54-64-8) one; C IO H 16 0 = 152.2 (546-80-5)
A light, yellowish white, crystalline powder; melting point, d~g, about 0.925; n~, about 1.455; [a]~, about -15; boiling
about 233°, with decomposition. point, about 86°.
Thiourea CH4 N 2S = 76.12 (62-56-6) Thymidine 1-(2-Deoxy-~-D-erythro-pentofuranosyl)-5-
Analytical reagent grade of commerce. methylpyrimidine-2,4(IH,3H)-dione; CIOH14N205 = 242 .2.
Threonine See L-Threonine. Thymine 5-Methylpyrimidine-2,4(IH,3H)-dione;
C SH 6N 20 2 = 126.1 (65-71-4)
L-Threonine C~9N03 = 119.1 (72-19-5)
Thyminose See 2-DeoXY-D-ribose.
A white, crystalline powder; [a]~, about -28 (2% w/v in
water) . Thymol 2-Isopropyl-5-methylphenol;
C IOH 14 0 = 150.2 (89-83-8)
Homogeneity Carry out the method for thin-layer General reagent grade of commerce.
chromatography, Appendix III A, protected from light, using Colourless crystals; melting point, about 50°.
s¡Jica gel G as the coating substance and a mixture of 25 Thymol used in gas chromatography complies with the following
parts of water and 75 parts of phenol as the mobile phase but test.
allowing the solvent front to ascend 12 cm from the line of Assay Examine by the method for gas chromatography in
application. Apply to the plate 5 ¡<L of a 0.2% w/v solution the monograph for Peppermint Oil using the reagent being
of the reagent being examined and keep the plate in the examined as the test solution. In the ehromatogram
vapour of the mobile phase for 12 hours before development. obtained, the area of the principal peak is not less than
After removal of the plate, dry it, spray with a 0.1 % w/v 95.0% by normalisation.
solution of ninhydrin in butan-1-o1 saturated with water and
0 0
heat at 100 to 105 for 10 minutes. The chromatogram
shows only one spot.
Thymol Blue Thymolsulfonphthalein; 4,4'-(3H)-2,1- Tin(n) Ch10ride Solution Rl Stannous chloride

Benzoxathiol-3-ylidene)dithymol S,S-dioxide; solution Rl
C 27 H 300 5S = 466.6 (76-61-9) lmmediately before use, dilute 1 volume of tin(n) chloride
A brownish green ro greenish blue crystalline powder. solution with 10 volumes of 2M hydrochloric acid.
Thymol Blue Solution Dissolve 0.1 g of thymol blue in Tin(n) Ch10ride Solution R2 Stannous chloride
2.15 mL of O.IM sodium hydroxide and 20 mL of ethanol solution R2
(96%) and add sufficient water ro produce 100 mL. To 8 g of tin(n) chloride add 100 mL of a 20% v/v solution of
C0111plies with the following tests. hydrochlorie acid. Shake until dissolved, heating, if necessary,
Sensitivity A mixture of 0.1 mL and 100 mL of carbon on a water bath at 50°. Pass a current of nitrogen through the
dioxide-free water ro which 0.2 mL of 0.02M sodium hydroxide solution for 15 minutes.
VS has been added is blue. Not more than 0.1 mL of Prepare irnmediately before use.
0.02M hydrochloric acid VS is required to change the colour of Titan Yellow Cl 19540; thiazol yellow;
the solution to yellow. CZSH19N5Naz06S4 = 696 (1829-00-1)
Colour change pH l.2 (red) to pH 2.8 (yellow); pH 8.0 A yellowish brown powder.
(olive green) ro pH 9.6 (blue). Titan Yellow Paper Impregnate filter paper with titan
Thymolphthalein 3,3-Bis(4-hydroxy-5-isopropyl-2- yellow solution. Allow to dry at room temperature.
methylphenyl)phthalide; CZSH3004 = 430.5 (125-20-2) Titan Yellow Solution A 0.05% w/v solution of titan
Produces a colourless solution in acidic and weakly alkaline yellow.
solutions and a blue colour in more strongly alkaline Complies with the following test.
solutions.
Sensitivity To 0.1 mL add 10 mL of water, 0.2 mL of
Thymolphthalein Solution A 0.1 % w/v solution of magnesium standard solution (10 ppm Mg) and l.0 mL of
thY1110lphthalein in ethanol (96%) . 1M sodium hydroxide. A distinct pink colour is visible by
C0111plies with the following tests. comparison with a reference solution prepared in the same
Sensitivity A mixture of 0.2 mL and 100 mL of carbon manner but omitting the magnesium solution.
dioxide-free water is colourless. Not more than 0.05 mL of Titanium Ti = 47.9 (7440-32-6)
O.IM sodiu111 hydroxide VS is required to change the colour of Metal powder, fine wire (diameter not more than 0.5 mm) or
the solution to blue. sponge containing not les s than 99% ofTi.
Colour change pH 9.3 (colourless) to pH 10.5 (blue). Melting point, about 1668°; densiry, 4.507 g cm-3.
Tin Granulated tin; Sn = 118.7 (7440-31-5) Titanium(m) Ch10ride Titanium trichloride;
Analytical granulated grade of commerce complying with the TiCl3 = 154.3 (7705-07-9)
following test. General reagent grade of commerce.
Arsenic 0.1 g complies with li111it test A for arsenie, Reddish violet crystals; melting point, about 440 0 •
Appendix VII (lO ppm).
Tin(u) Ch10ride Stannous chloride;
SnClz,2H zO = 225 .6 (10025-69-1) Titanium(m) Ch10ride Solution Titanium trichloride
solution
Analytical reagent grade of commerce containing not less
than 97.0% of SnClz,2HzO. General reagent grade of commerce containing about
15% w/v ofTiCl3 in hydroehlorie acid (10% w/v HCI).
Assay Dissolve 0.5 g in 15 mL of hydroehlorie acid in a
ground-glass-stoppered flask and add 10 mL of water and Weight per mL, about l.2 g.
5 mL of ehloroform. Titrate rapidly with 0.05M potassium Titanium(m) Ch1oride-Sulfuric Acid Reagent
iodate VS until the chloroform layer is colourless. Each mL of Titanium(m) chloride-sulphuric acid reagent, titanium
0.05M potassiu111 iodate VS is equivalent ro 22.56 mg of trichloride- sulfuric acid reagent, titanium trichloride-
SnClz,2H zO. sulphuric acid reagent
Tin(u) Ch10ride Solution Stannous chloride solution Carefully mix 20 mL of titanium(m) ehloride solution with
Heat 20 g of tin with 85 mL of hydroehlorie acid until no 13 mL of sulfurie acid, add sufficient hydrogen peroxide solution
(100 voD to produce a yellow colour, heat until white fumes
more hydrogen is evolved.
are evolved and allow ro cool. Dilute with water and repeat
Store over an excess of tin and protected from airo the evaporation and addition of water until a colourless
Tin(u) Ch10ride Solution AsT solution is obtained. Dilute this solution to 100 mL with
Stannous chloride solution, low in arsenic, of commerce, or water.
prepare from tin(n) ehloride solution by adding an equal Titanium Dioxide Titanium(rv) oxide;
volume of hydroehlorie acid, reducing the original volume by TiO z = 79.90 (13463-67-7)
boiling and filtering through a fine-grain filter paper. General reagent grade of commerce.
C0111plies with the following test. Titanium Trichloride See Titanium(w) ehloride.
T o 10 mL add 6 mL of water and 10 mL of hydroehloric acid, Titanium Trich10ride Solution See Titanium(w) ehloride
distil and collect 16 mL. To the distillate add 50 mL of solution.
water, 0.1 mL of the solution, 5 mL of O.IM potassiu111 iodide
and 5 g of activated zinc. Using the apparatus and procedure Titanium Trichloride-Sulfuric Acid Reagent See
described for the li111it test for arsenie, Appendix VII, the stain Titanium(w) chloride- sulfurie aeid reagent.
produced on 111ercuric bromide paper is not more intense than TLCAluminium Oxide G Plate
that produced when the test is repeated with the addition of Suppott of metal, glass or plastic, coated with a layer of
1 mL of arsenic standard solution (1 ppm As). aluminium oxide (particle size 5 to 40 11m) containing about
10 % of calcium sulfate hemihydrate as a binder.
TLC Octadecylsilyl Silica Gel Plate Suppon of glass, Storage protected from light, in an open container; use within
metal or plastic coated with a layer of octadecylsilyl silica gel. 30 days after preparation .
The plate may contain an organic binder. TLC Silica Gel Plate for Chiral Separations,
TLC Octadecylsilyl Silica Gel F 254 Plate Suppon of Octadecylsilyl
glass, metal or plastic coated with a layer of octadecylsilyl Suppon of glass, metal or plastic, coated with a layer of
silica gel. octadecylsilyl silica gel, impregnated with Cu 2 + ions and
It contains a fiuorescent indicator having a maximum enantiomerically pure hydroxyproline. The plate may contain
absorbance in ultraviolet light at 254 nm. an organic binder.
TLC Performance Test Solution Prepare a mixture of TLC Silica Gel Silanised Plate
1 mL of each of the following solutions and dilute to 10 mL Suppon of glass, metal or plastic, coated with a layer of
with acetone: a 0.05% w/v solution of sudan red G in toluene, silanised silica gel of a suitable thickness and particle size
a 0.05 % w/v solution of methyl orange in absolute ethanol (usually 2 to 10 pm for fine particle size (high performance
prepared immediately before use, a 0.05% w/v solution of thin layer chromatography, HPTLC, plates) and 5 to 40 pm
bromoeresol green in aeetone and a 0.025% w/v solution of for normal plates). If necessary, the particle size is indicated
methyl red in aeetone. after the name of the reagent in the tests where it is used.
TLC Silica Gel Plate Silica gel An organic binder may be inc1uded.
Suppon of glass, metal or plastic, coated with a layer of silica Chromatographic separation Introduce 0.1 g each of
gel of a suitabl~ thickness and particle size (usually 2 to 10 methyl laura te, methyl myristate, methyl palmitate and methyl
~lm for fine particle size (high performance thin layer stearate into a 250 mL conical fiask, add 40 mL of alcoholic
chromatography, HPTLC, plates) and 5 to 40 pm for normal potassium hydroxide solution and heat under a refiux condenser
pi ates) . If necessary, the particle size is indicated after the on a water bath for 1 hour. Allow 10 cool, transfer the
name of the reagent in the tests where it is used. An organic solution to a separating funnel with the aid of 100 mL of
binder may be inc1uded. water, acidify to pH 2 10 3 with 2M hydroehloric aeid and
TLC Silica Gel F254 Plate extract with three 10-mL quantities of diehloromethane. Dry
Complies with the requirements prescribed for TLC siliea gel the combined extracts over anhydrous sodium sulfate, filter and
plate with the following modifications. evaporate to dryness on a water bath. Dissolve the residue in
It contains a fiuorescent indicator having a maximum 50 mL of diehloromethane. Examine by thin-layer
absorbance at 254 nm. ehromatography, Appendix III A, using a TLC silica gel
silanised plate and a mixture of 10 volumes of glacial acetie
Fluorescence suppression Apply separately to the plate
acid, 25 volumes of water and 65 volumes of 1,4-dioxan as
at five points increasing volumes (1 pL to 10 pL for normal
the mobile phase but allowing the solvent front to ascend two
pi ates and 0.2 pL to 2 pL for fine particle size plates) of a
thirds of the height of the plate. Apply separately to the plate
0.1 % w/v solution of benzoie aeid in a mixture of 15 volumes
an appropriate quantity (about 10 pL of normal plates and
of absolute ethanol and 85 volumes of eyclohexane. Develop
about 1 pL 10 2 pL for fine partic1e size plates) of the
over a pathlength of half the plate height using the same
dichloromethane solution at each of three separate points.
mixture of solvents as the mobile phase . After removal of the
After removal of the plate, dry it at 120° for 30 minutes,
plate, allow the solvents to evaporate and examine the
allow to cool, spray with a 3.5% w/v solution of
chromatograms in ultraviolet light (254 nm). For normal
phosphomolybdie acid in propan-2-o1 and heat at 150° until the
TLC plates benzoic acid appears as dark spots on a
spots become visible. Treat the plate with ammonia vapour
fiuorescent background approximately in the middle of the
until the background is white. The chromatograms show four
chromatogram for quantities of 2 pg or greater. For fine
c1early separated, well-defined principal spots.
particle size plates benzoic acid appears as dark spots on a
fiuorescent background approximately in the middle of the IX-Tocopherol (10191-41-0)
chromatogram for quantities of 0.2 ~lg or greater. See all-rac-IX-Toeopherol of the British Pharmacopoeia.
TLC Silica Gel F 254 Silanised Plate IX-Tocopheryl Acetate (7695-91-2)
Complies with the requirements prescribed for TLC siliea gel See all-rac-IX-Toeopheryl Aeetate of the British Pharmacopoeia.
silanised plate with the following modifications. 0- Tolidine 3,3'-Dimethylbenzidine;
It contains a fiuorescent indicator having a maximum C14H16N2 = 212.3 (119-93-7)
absorbance at 254 nm.
TLC Silica Gel G Plate
97.0% of C1 4H16NZ.
Complies with the requirements prescribed for TLC siliea gel
plate with the following modifications.
0- Tolidine Solution Dissolve 0.16 g of o-tolidine in
It contains ca1cium sulfate hemihydrate as a binder.
30 mL of glacial aeetie acid, add 1 g of potassium iodide and
TLC Silica Gel GFZ54 Plate
Complies with the requirements prescribed for TLC siliea gel
Toluene Methylbenzene; C 7 H s = 92.14 (108-88-3)
plate with the following modifications.
It contains ca1cium sulfate hemihydrate as binder and a Analytical reagent grade of commerce.
fiuorescent indicator having a maximum absorbance at A colourless liquid; weight per mL, 0.865 10 0.870 g; boiling
254 nm. point, about 110°.
Fluorescence suppression Complies with the test Toluene, Sulfur-free Toluene, sulphur-free
prescribed for TLC siliea gel F254 plateo Toluene that complies with the following tests.
TLC Silica Gel Plate for Aminopolyether Test Sulfur compounds To 10 mL add 1 mL of absolute
Immerse a TLC siliea gel plate in iodoplatinate reagent R1 for ethanol and 3 mL of potassiwn plumbite solution and boil
5-10 s. Dry at room temperature for 12 h, protected from under a refiux condenser for 15 minutes. Allow to stand for
light. 15 minutes; no darkening is produced in the aqueous layer.
Thiophene-related substances Shake 2 mL with 5 mL Tosylarginine Methyl Ester Hydrochloride Methyl

of isatin reagent for 5 minutes and allow to stand for N-tosyl-L-argininate hydrochloride;
15 minutes. No blue colour is produced in the lower layer. C1 4HzzN40 4S,HCI = 378.9 (1784-03-8)
To1uenesulfonamide See Toluene-p-sulfonamide. Use a grade of commerce suitable for the assay of trypsin.
0- Toluenesulfonamide See Toluene-o-sulfonamide. A white, crystalline powder; melting point, about 145°;
p- Toluenesulfonamide See Toluene-p-sulfonamide. [al~, - 12 to - 16 (4% w/v in water).
Toluene-o-sulfonamide Toluene-o-sulphonamide; 0- T osylarginine Methyl Ester Hydrochloride Solution
Toluenesulfonamide; 0- Toluenesulphonamide; 2- Dissolve 98.5 mg of tosylarginine methyl ester hydroehloride in
methylbenzenesulfonamide; 2-methylbenzenesulphonamide; 5 mL of tris-ehloride buffer, pH 8.1, shaking to effect
C 7H gNO zS = 171.2 (88-19-7) dissolution, add 2.5 mL of methyl red mixed solutwn and
General reagent grade of commerce. dilute to 25 mL with water.
Melting point, about 156°. Tosyl-lysyl-ch1oromethane Hydroch1oride (3S)-7-
Amino-1-chloro-3-tosylamido-2-heptanone hydrochloride; N-
Toluene-p-sulfonamide Toluene-p-sulphonamide; p- tosyl-L-Iysy1chloromethane hydrochloride;
toluenesulfonamide, p-toluenesulphonamide; toluene C14HzI CINz03S,HCI = 369.3 (4238-41-9)
sulfonamide, toluene sulphonamide; 4-
methylbenzenesulfonamide, 4-methylbenzenesulphonamide; General reagent grade of commerce.
C 7H g N OzS = 171.2 (70-55-3) Melting point, about 155°, with decomposition;
General reagent grade of commerce. [albo, - 7 to - 9 (2% w/v); A(l %, 1 cm) at 230 nm, 310 to
340 (water) .
Tosylphenylalanylch1oromethane N- Tosyl-L-
Complies with the following test. phenylalany1chloromethane; L-tosylaminophenethyl
Homogeneity Carry out the test for Related substances chloromethyl ketone; C 17H 18 CIN0 3S = 351.9 (402-71-1)
described in the monograph for Tolbutamide applying to the General reagent grade of commerce.
plate 5 IlL of a 0.015% w/v solution in aeetone. The
chromatogram shows only one principal spot. A white, crystalline solid, melting point, about 105°;
[albo, - 85 to - 89 [1 % w/v in ethanol (96%)1; A(l %, 1 cm),
Toluenesulfonic Acid See Toluene-p-sulfonie aeid. 290 to 320 determined at 228.5 nm in ethanol (96%).
Toluene-p-sulfonic Acid Toluene-p-sulphonic acid; Toxaphene (8001-35-2)
toluenesulfonic acid, toluenesulphonic acid; 4-
methylbenzenesulfonic acid, 4-methylbenzenesulphonic acid; A mixture of polychloro derivatives.
C 7H s0 3S,H zO = 190.2 (6192-52-5) Use a grade of commerce suitable for pesticide residue
analysis. A suitable certified reference solution (10 ng/llL in
iso-octane) may be used.
87.0% of C 7H s0 3 S.
A white, crystalline powder or crystals. Melting point, 65° to 90°.
Tragacanth (9000-65-1)
To1uenesulfonylurea 4-Methylbenzenesulfonylurea; 4-
methylbenzenesulphonylurea, p-toluenesulfonylurea; p- Of the British Pharmacopoeia.
toluenesulphonylurea, (4-methylphenyl)sulfonylurea; Triacetin Propane-1,2,3-triyl triacetate;
(4-methylphenyl)sulphonylurea; C 9 H 14 0 6 = 218.2 (102-76-1)
C SHION z0 3S = 214.2 (1694-06-0) General reagent grade of commerce.
White or almost white, crystalline powder; melting point, d~g, about 1.16; n~, about 1.43; boiling point, about 260 0
•
196° to 198°. Triamcinolone C z1 H 27 F0 6 = 394.4 (124-94-7)

o-Toluic Acid C8H80Z = 136.2 (118-90-1) General reagent grade of commerce.
General reagent grade of commerce. Melting point, 262° 10 263°.
A white, crystalline powder; melting point, about 104°. Triamcinolone Acetonide C Z ~31F 0 6 = 434.5 (76-25-5)
o-Toluidine 2-Methylaniline; C 7H gN = 107.2 (95-53-4) General reagent grade of cornmerce.
General reagent grade of commerce. T ribromophenol 2,4,6-Tribromophenol;
A pale yellow liquid becoming reddish-brown on exposure to C6H3Br30 = 330.8 (118-79-6)
air and light; d~g, about 1.01; n~, about 1.569; boiling poínt, Tributyl Citrate Tributyl 2-hydroxypropane-1,2,3-
about 200°. tricarboxylate; C18H3Z07 = 360.4 (77-94-1)
Store in an airtight container protected from light. d¡O, about 1.043; nbo, about 1.445.
p- Toluidine 4-Methylaniline; C 7H gN = 107 .2 (106-49-0) Tributyl Orthophosphate Tributyl phosphate;
General reagent grade of commerce. ClzHz704P = 266 .3 (126-73-8)
Lustrous plates or f1akes; melting point, about 44°. General reagent grade of commerce washed before use with
Toluidine Blue Toluidine blue O; CI 52040; 3-amino-7- three quantities, each of one-sixth of its volume, of a solution
dimethy1amino-2-methylphenothiazin-5-iurn chloride; containing 10% w/v of sodium ehloride and 1.0% w/v of
ClsH16CIN3S = 305.8 (92-31-9) disodium hydrogen orthophosphate.
A colourless liquiq; weight per mL, about 0.98 g.
Tributyl Phosphate Tributoxyphosphine oxide,
A dark green powder.
tributoxyphosphane oxide; ClzHz704P = 266.3 (126-73-8)
0- Toluidine Hydrochloride 2-Methylaniline Colourless liquid, slightly soluble in water, soluble in the
hydrochloride; C 7H g N,HCI = 143.6 (636-21-5) usual organic solvents.
General reagent grade of commerce. d~~, about 0.976; nf], about 1.422.
Melting point, 215° to 217°. Boiling point, about 289°, with decomposition.
Trichlorethylene See Trichloroethylene. Water, maximum 0.2%.

Trichloroacetic Acid C 2HCI 30 Z = 163.4 (76-03-9) It is suitable for gradient elution in liquid chromatography.
Analytical reagent grade of commerce. Use freshly distilled or from a freshly opened container.
Colourless, deliquescent crystals . Triethylamine Hydrogen Carbonate Solution Pass a
Store in an airtight container. gentle current of carbon dioxide through a 5% w/v solution of
tn'ethylamine for 16 hours.
Trichloroacetic Acid Solution Dissolve 40 g of
trichloroacetic acid in sufficient water to produce 1000 mL. Triethylenediamine 1,4-DiazobicycIo[2.2.2]octane;
Verify the concentration by titration with O.IM sodium C 6H 1ZN z = 112.2 (280-57-9)
hydroxide VS and adjust, if necessary, to 40 ± 1 gIL. General reagent grade of commerce.
1,1,1-Trichloroethane MethyIchloroform; Hygroscopic crystals which sublime readily at room
CZH3CI3 = 133.4 (71-55-6) temperature; melting point, about 158°; boiling point, about
General reagent grade of commerce. 174°.
d~g, about 1.34; n~, about 1.438; boiling point, about 74°. Store in an airtight container.
Trichloroethylene Trichlorethylene; Triflumuron 1-(2-Chlorobenzoyl)-3-
C zHCI 3 = 131.4 (79-01-6) (4-trifiumoromethoxyphenyl)urea;
Technical grade of commerce. CI sHIOCIF3N203 = 358.7 (64628-44-0)
White or almost white crystalline powder, practically
Colourless liquid; d~g, about 1.46; n~, about 1.477.
insoluble in water, sparingly soluble in acetone and in
Trichlorotrifluoroethane 1,1,2-Trichlorotrifiuoroethane; dichloromethane .
CZCI3F3 = 187.4 (76-13-1)
Trifluoroacetic Acid C2HF30 2 = 114.0 (76-05-1)
Reagent grade of commerce suitable for protein sequencing
A colourIess, volatile liquid; boiling point, about 47°; d~g, containing not less than 99% of C 2H0 2F 3.
about 1.58.
Boiling point, about 72 0 ; d~g, about 1.53.
Distillation range Not less than 98% distils between 47°
and 48°, Appendix V C. Trifluoroacetic Anhydride C 4 F 60 3 = 210.0 (407-25-0)
Tricine N- [2-Hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine; General reagent grade of commerce.
C6H13NOs = 179.2 (5704-04-1) A colourless liquid; d~g, about 1.5.
Use electrophoresis-grade reagent. 3-Trifluoromethylaniline 3-(Trifiuoromethyl)aniline;
Melting point, about 183°. a,a,a-Trifiuoro-m- toluidine;
3-(Trifiuoromethyl)benzenamide;
Tricosane C z3 H 48 = 324.6 (638-67-5) C7H6F3N = 161.1 (98-16-8)
General reagent grade of commerce. ColourIess liquid; density: 1.30 g/cm 3 (20°).
White crystals, practically insoluble in water, soluble in 4-Trifluoromethylphenol C7HsF30 = 162.1 (402-45-9)
hexane; melting point, about 48°.
White or light yellow, crystalline solid or powder.
Tridocosahexaenoin Triglyceride of docosahexaenoic acid
(C22:6); Glycerol tridocosahexaenoate; Propane-l,2,3-triyl Melting point, about 46°.
tri-(all-Z)-docosa-4, 7,10,13,16, 19-hexaenoate; Trigonelline Hydrochloride l-Methylpyridinium-3-
C69H9806 = 1023.5 (124596-98-1) carboxylate hydrochloride;
The reagent from Nu-Chek Prep, Inc. has been found C 7H 7NO z,HCI = 173.6 (6138-41-6)
suitable. General reagent grade of commerce.
0
Triethano1amine C 6H 1S N0 3 = 149.2 (102-71-6) Melting point, about 258 •
General reagent grade of commerce. Trimethy1chlorosilane Chlorotrimethylsilane;

A viscous, very hygroscopic, colourless liquid; d~g, about C 3H 9 CISi = 108.7 (75-77-4)
1.13. General reagent grade of commerce.
Store in an airtight container protected from light. A colourIess liquid; d~g, about 0.86; n~, about 1.388; boiling
Triethyl Phosphonoformate Ethyl point, about 57°. '
(diethoxyphosphoryl)formate; C 7H 1S OsP = 210.2 (1474- Trimethylpentane See 2,2,4-Trimethylpentane.
78-8) 2,2,4-Trimethylpentane Iso-octane; trimethylpentane;
General reagent grade of commerce. C SH I8 = 114.2 (540-84-1)
n~, about 1.423. General reagent grade of commerce.
Triethylamine C 6H 1S N = 101.2 (121-44-8) A colourless, fiammable liquid; d~g, 0.691 to 0.696; n~o,
General reagent grade of commerce. 1.391 to 1.393 .
A colourIess liquid; d~g, about 0.727; n~, about 1.401;
boiling point, about 90°. Distillation range Not less than 95% distils between 98°
0
Triethylamine R2 N,N- Diethylethanamine; and 100 •
C 6H 1SN = 101.2 (121-44-8) 2,2,4-Trimethylpentane used in spectrophotometry complies with

Complies with the requirements prescribed for niethylamine
and with the following additional requirements. Transmittance Not les s than 98% between 250 and
Content, minimum 99.5%, determined by gas 420 nm, Appendix II B, using water in the reference cel!.
chromatography.
Trimethylpentane for chromatography Complies with Triphenyltetrazolium Chloride Solution A 0.5% w/v
the requirements prescribed for trimethylpentane with the solution of 2,3,5-niphenyltetrazolium ehloride in aldehyde-free
following additional requirement. ethanol (96%).
Residue on evaporation Maximum 2 mg!L. Store protected from light.
Trimethylpentane Rl 2,2,4-Trimethylpentane complying Tripotassium Phosphate Trihydrate
with the following test. K 3P0 4 ,3H20 = 266.3 (22763-03-70)
Absorbance Not more than 0.07 from 220 nm to 360 nm General reagent grade of commerce.
determined using water in the reference cel!. T riscyanoethoxypropane
N-Trimethylsilylimidazole C 6H 12 N 2Si = 140.3 (18156- 1,2,3-Tris(cyanoethoxy)propane; C12H17N30 3 = 251.3
74-6) Chromatographic reagent grade of commerce.
Analytical reagent grade of commerce. A viscous, brownish yellow liquid; dig, about 1.11; viscosity,
A colourless, hygroscopic Iiquid; dig, about 0.96; n~, about about 172 mPa·s, Appendix V H, Method 11.
1.48. 1,3,5 -Tris(3,5-di-(I, l-dimethylethyl)-4-hydroxybenzyl)-
Store in an airtight container. IH,3H, 5H-I ,3, 5-triazine-2 ,4,6-trione T ris(3 ,5-di-tert-
3-Trimethylsilyl-l-propanesulfonic Acid, Sodium Salt butyl-4-hydroxybenzyl isocyanurate;
Sodium 3-(trimethylsilyl)-l-propanesulfonate; sodium C4sH 69N 30 6 = 784.1 (27676-62-6)
3-(trimethylsilyl)-I-propanesulphonate; 3-trimethylsilyl-l - General reagent grade of commerce.
propanesulphonic acid, sodium salt; Melting point, 218° to 222°.
C6H1 5Na03SSi = 218.3 (2039-96-5) Tris(2,4-di- (1, l-dimethylethyl)phenyl) Phosphite
Beige powder; content, minimum 97 .0 %; melting point, C42H 630 3P = 647 (3 1570-04-4)
about 165°. General reagent grade of cornmerce.
Trimethylsulfonium Hydroxide Trimethylsulfonium Melting point, 182 0 to 186°.
hydroxide; C 3H lO OS = 94.2 (17287-03-5)
Tris(hydroxymethyl)nitromethane
d¡O, about 0.81. C 4 H gN0 5 = 151 (126-11-4)
2,4,6-Trinitrobenzenesulfonic Acid Picrylsulfonic acid, General reagent of commerce.
picrylsulphonic acid, 2,4,6-trinitrobenzenesulphonic acid;
Tris (hydroxymethylaminomethane) See
C6H3N 30 9S,3H20 = 347.2 (2508-19-2)
Tris (hydroxymethyl) methylamine.
Tris(hydroxymethylaminomethane) Solution See
Melting point, 190° to 195°. Tris (hydroxymethyl) methylamine solution.
Triolein. Propane-l,2,3-triyl tris [(9Z)-octadec-9-enoate1; Tris(hydroxymethyl)aminomethane Solution RI
sn-Glyceryl trioleate; Glycerol trioleate; Oleyl triglyceride; Dissolve 60.6 mg of tris(hydroxymethyl)methylamine and
C57H10406 = 885.4 (122-32-7) 0.234 g of sodium ehloride in sufficient water to produce
Content, minimum 99.0%. 100 mL.
Triphenylamine C 1sH 15N = 245.3 (603-34-9) Store at 2° to 8° and use within 3 days .
General reagent grade of commerce. Tris(hydroxymethyl)methylaniine
A white, crystalline solid; melting point, about 126°. Tris(hydroxymethyl)aminomethane; trometamol;
Triphenylethylene C 2o H 16 = 256.4 (58-72-0) C 4H 11 N0 3 = 12l.l (77-86-1)
General reagent grade of commerce. Analytical reagent grade of commerce.
Triphenylmethanol Triphenylcarbinol; Tris(hydroxymethyl)methylamine Solution
C l9 H l60 = 260.3 (76-84-6) Tris(hydroxymethyl)aminoethane solution
General reagent grade of commerce. A 2.422% w/v solution of tris(hydroxymethyl)methylamine
(O.IM).
Triphenyltetrazolium Chloride See
2,3,5- Triphenyltetrazolium ehloride. Tris(hydroxymethyl)methylamine Solution, Methanolic
2,3,5-Triphenyltetrazolium Chloride Tetrazolium salt; Dissolve 6.07 g of tris(hydroxymethyl)methylamine in 900 mL
of methanol, add 50 mL of 0.5M hydroehlorie aeid and dilute
Cl 9H15CIN4 = 334.8 (298-96-4)
to 1000 mL with water.
Trisodium Orthophosphate Trisodium phosphate
than 98.0% of C19H15CIN4'
dodecahydrate; Na3P04,12H20 = 380.1 (10101-89-0)
A pale yellow or cream powder; melting point, about 240°,
with decomposition.
Trisodium Phosphate Dodecahydrate See Trisodium
Assay Dissolve 1 g in a mixture of 5 mL of dilute nitrie
orthophosphate.
aeid and 45 mL of water, add 50 mL of O.IM si/ver nitrate VS
and heat to boiling. Allow to cool, add 3 mL of dibutyl Tropic Acid (2RS)- 3-hydroxy-2-phenylpropanoic acid;
phthalate, shake vigorously and titrate with O.IM ammonium C9HJQ03 = 166.17; (529-64-6)
thioeyanate VS using 2 mL of ammonium iron(J/l) sulfate Tropine (IR,3r,5S)- Tropan-3-ol;
solution R2 as indicator. Each mL of 0.1 M silver nitrate VS is C S H 15NO = 141.2 (120-29-6)
equivalent to 33.48 mg of ClgH15CIN4. General reagent grade of commerce.
Store protected from light. Colourless crystals; melting point, about 54°.
Troxerutin Trihydroxyethylrutin; 3',4',7-Tris[O-
(2-hydroxyethyl) 1rutin. 2- [3 ,4-Bis (2-hydroxyethoxy)phenyl]-
3-[[6-0-(6-deoxY-IX-L-mannopyranosyl)-P-D- Valencene 4 PH, 51X-Eremophila-l (lO),II-diene.

glucopyranosyl) oxy)-5-hydroxy-7 -(2-hydroxyethoxy)-4H-l- (IR, 7R,8aS)-1 ,8a-DimeIhyl-7 -( l-meIhylethenyl)-
benzopyran-4-one; C33H4201 9 = 743 (7085-55-4) 1,2,3,5,6,7,8,8a-octahydronaphIhalene;
Melting point, 168° to 176°. C 1sH 24 = 204.4 (4630-07-3)
Trypsin (9002-07-7) A proteolytic enzyrne obtained by Oily, colourless or pale yellow liquid, with a characteristic
activation of trypsinogen extracted from the pancreas of odour, practically insoluble in water, soluble in ethanol
cattle (Eos taurus L.). (96%).
A white, crystalline or amorphous powder. d¡O, about 0.918; nt>, about 1.508; boiling point, about
123°.
Trypsin for Peptide Mapping
Trypsin of high purity treated to eliminate chymotryptic
Valencene used in gas chromatography complies with the following
activity. additional test.
Tryptophan CIIHI2N202 = 204.2 (73-22-3)
Assay Examine by gas chromatography (2.2.28) as
prescribed in the monograph on Sweet orange oi!.
The content is not less Ihan 80%, calculated by Ihe
[a)~o, about -30°, determined in a 1% w/v solution.
Tyrarnine 4-(2-Aminoethyl)phenol;
Valerenic Acid (2E)-3-[(4S,7R,7aR)-3,7-DimeIhyl-
CsH11NO = 137 .2 (51-67-2) 2,4,5,6,7,7 a-hexahydro-1H-inden-4-yl] -2-meIhylprop-2-enoic
General reagent grade of commerce. acid; CIsH2202 = 234.3 (3569-10-6)
Melting point, 164° to 165°. Melting point, 134 0 to 138°.
Tyrosine See L-Tyrosine. Valeric Acid n-Valeric acid; pentanoic acid;
L-Tyrosine 2-Amino-3-(4-hydroxyphenyl)propionic acid; C S H IO 0 2 = 102.1 (109-52-4)
tyrosine; C 9H II N0 3 = 181.2 (60-18-4) Colourless liquid, soluble in water, freely soluble in eIhanol
General reagent grade of commerce complying with the (96%).
following test. dgg, about 0.94; n~o, about 1.409; boiling point, about 186°.
Homogeneity Carry out the test for Related substances in Valine Of the British Pharmacopoeia
the monograph for Levodopa. The chromatogram shows Vanillin 4-Hydroxy-3-methoxybenzaldehyde;
only one spot.
C SH S0 3 = 152.2 (121-33-5)
Umbelliferone 7-Hydroxycoumarin; 7-hydroxy-2H-l-
benzopyran-2-one; C9Hó03 = 162.1 (93-35-6)
Vanillin Reagent Carefully add, dropwise, 2 mL of sulfuric
Needles from water. acid to 100 mL of a 1 % w/v solution of vani!lin in ethanol
Melting point, 225° to 228°. (96%).
Undecanoic Acid C 11 H 22 0 2 = 186.3 (112-37-8) Use within 48 hours.
General reagent grade of commerce. Vanillin Solution, Phosphoric Dissolve 1.0 g of vanillin
Melting point, about 28.5°. in 25 mL of ethanol (96%). Add 25 mL of water and 35 mL
Uracil C 4H 4N 20 2 = 112.1 (66-22-8) of phosphoric acid.
Content, minimum 95.0%. Veratric Acid 3,4-Dimethoxybenzoic acid;
C 9 H IO 0 4 = 182.2 (93-07-2)
Urea CH 4N 20 = 60.06 (57-13-6)
0
Urease-active Meal (9002-13-5)
General reagent grade of commerce. Veratrole 1,2-Dimethoxybenzene;
C SH IO 0 2 = 138.2 (91-16-7)
Activity Each mg of urease-active meal hydrolyses 3 mg of
urea in 30 minutes at 37°. d¡O, 1.085; nt>, 1.534; boiling point, about 206°; melting
point, about 22°.
Uridine l-P-D-Ribofuranosyluracil;
C 9 H 12 N 20 ó = 244.2 (58-96-8) Verbenone (lS,5S)-4,6,6-TrimeIhylbicyclo[3 .1. 1.)hept-3-
en-2-one; C 1oH I40 = 150.2 (1196-01-6)
White or almost white, crystalline powder, soluble in water.
Oil with a characteristic odour, practically insoluble in water,
miscible with organic solvents.
Ursolic Acid 3P-Hydroxyurs-12-en-28-oic acid;
dgg, about 0.978; nb8, about 1.49; [albs, about + 249 .6;
C30H4S03 = 456.7 (77-52-1) boiling point, 227° to 228°; melting point, about 6.5°.
White or almost white powder, practically insoluble in water, Verbenone used in gas chromatography complies with the following
sparingly soluble in methanol, slightly soluble in ethanol additional test.
(96%).
[a)ii, about + 67.50, determined on a 10 gIL solution in a monograph Rosemary oi! (1846).
56.1 gIL solution of potassium hydroxide in ethanol (96%).
Content, minimum 99%, calculated by Ihe normalisation
procedure.
UrushiolI 3-Pentadecyl-l,2-benzenediol; C21H3ó02 VinyI Acetate EIhenyl acetate; C 4H 6 0 2 = 86.1 (108-05-4)
= 320.51 (492-89-7)
dgg, about 0.930; boiling point, about 72°.
General ragent grade of commerce.
VinyI Chloride C 2H 3CI = 62.5 (75-01-4)
UrushioI II 3-(8-Pentadecenyl)-1,2-benzenediol;
Colourless gas, slightly soluble in organic solvents .
C21H3402 = 318.26 (2764-91-2)
General ragent grade of commerce.
Vinyl Polymer for Chrornatography, Octadecyl Water, Arnrnoniurn-free See Water, ammonia-free.
Spherical particles (5 ~lm) of a vinyl alcohol copolymer Water, Carbon Dioxide-free Water which has been
chemically modified by bonding of octadecyl groups on the boiled for a few minutes and protected from the atmosphere
hydroxyl groups. during cooling and srorage.
Vinyl Polymer for Chrornatography, Octadecylsilyl Water, Distilled Purified Water of the British
Spherical particles (5 ~m) of a vinyl alcohol copolymer Pharrnacopoeia that has been prepared by distillation.
bonded ro an octadecylsilane. Carbon content of 17%. Water, Distilled, Deionised Deionised water prepared by
2-Vinylpyridine C7H7N = 105.1 (100-69-6) distillation with a resistivity of not less than 0.18 Mohm·m.
Yellow liquid, miscible in water. Water for Chrornatography Deionised water with a
resistivity of not less than 0.18 Mohm·m.
dig, about 0.97; nbo, about 1.549.
Water for Injections Of the British Pharrnacopoeia.
1-Vinylpyrrolidin-2-one 1-Ethenylpyrrolidin-2-one;
C 6 H 9 NO = 111.1 (88- 12-0) Water, Nitrate-free To 100 mL of water add a few
milligrams of potassium pennanganate and of barium hydroxide.
Distil using the apparatus described in Appendix V C. Reject
Clear colourless liquid. the first 10 mL and collect the following 50 mL.
Water (2.5.12) Maximum 0.1%, deterrnined on 2.5 g. Water, Partic1e-free
Use as the solvent, a mixture of 50 mL of anhydrous
Filter water through a membrane with a pore size of
methanol and 10 mL of butyrolactone.
0.22 ~m.
Assay Gas chromarography (2.2.28): use the normalisation
Xanthydrol 9-Hydroxyxanthene; xanthen-9-01;
procedure.
C I3H IO 0 2 = 198.2 (90-46-0)
Column:
- material: fused-silica,
White or pale-yellow powder, very slightly soluble in water,
- size: l = 30 m, 0 = 0.5 mm,
soluble in ethanol (96 %) and in glacial acetic acid.
- stationary phase: macrogol 20 000 R .
It is also available as a methanolic solution containing 90 giL
Camer gas helium for chromatography.
ro 110 gIL of xanthydro!.
Temperature:
Time Temperature Assay In a 250 mL fiask dissolve 0.300 g in 3 mL of
(min) (OC) methanol or use 3.0 mL of solution. Add 50 mL of glacial
Column 0-5 35 acetic acid and, dropwise with shaking, 25 mL of a 20 giL
5 - 15 35 - 85 solution of urea. Allow to stand for 12 hours, collect the
precipita te on a sintered-glass filter (16) (2.1.2), wash with
Injection port 250
20 mL of ethanol (96%); dry in an oven at 100° ro 1050 and
Detector 250 weigh.
1 g of precipitate is equivalent to 0.9429 g of xanthydro!.
Detection Flame ionisation. Store protected from light. If a methanolic solution is used,
Inject 0.3 ~L of the substance to be examined. store in small sealed ampoules and filter before use if
Adjust the fiow rate of the carrier gas so that the retention necessary.
time of the peak corresponding to l-vinylpyrrolidin-2-one is Xanthydrol Rl
about 17 minutes . Complies with the requirements prescribed for xamhydrol
Vitexin Apigenin-8-glucoside; with the following requirement.
C21H2001 0 = 448.4 (3681-93-4) Content, minimum 98.0% of C I3H IO 0 2.
Yellow powder. Xanthydrol Reagent Dissolve about 0.125 g of xanthydrol
Srore in an airtight container, protected from light. in 100 mL of anhydrous acetic acid. Add 1 mL of hydrochloric
Water (7732-18-5) Purified Water of the British acid immediately before use.
Pharrnacopoeia. Xanthydrol Solution To 0.1 mL of a 10% w/v solution of
Water Rl Prepared from distilled water by multiple xamhydrol in methanol add 100 mL of anhydrous acetic acid
distillation. Remove carbon dioxide by boiling for at least 15 and 1 mL of hydrochloric acid. Allow ro stand for 24 hours
min before use in a boiling fiask of fused silica or borosilicate before using.
glass and coo!. Any other suitable method m ay be used. Xylene A mixture of 0-, m- and p-isomers;
The boiling fiask has been already used for the test or has CsH IO = 106.2 (1330-20-7)
been filled with waterand kept in an autoclave at 121' for at Mixture of isomers . Clear, colourless, fiammable liquid,
least 1 h prior to first use. When tested immediately before practically insoluble in water, miscible with ethanol (96%) .
use, water Rl is neutral to methyl red solution, i.e. it shall dig, about 0.867; nbo, about 1.497; boiling point, about
produce an orange-red (not a violet-red or yellow) colour 138°:
corresponding to pH 5.5 ± 0.1 when 0.05 mL of methyl red m-Xylene 1,3-Dimethylbenzene; CsH IO = 106.2 (108-
solution is added to 50 mL of the water to be examined. 38-3)
Conductivity Maximum 1 ~S ·cm - 1, deterrnined at 25° by Clear, colourless, fiammable liquid, practically insoluble in
an in-line conductivity meter (se e Purified water (0008) . water, miscible with ethanol (96%).
Water, Arnmonia-free Ammonium-free water dig, about 0.884; n~, about 1.497; boiling point, about
To 100 mL of water add 0.1 mL of sulfuric acid (96% wlw). 139°; melting point, about _47°.
Distil using the apparatus described in Appendix V C. Reject
the first 10 mL and collect the following 50 mL.
o-Xylene 1,2-Dimethylbenzene; CSH 10 = 106.2 (95-47-6) Zinc Chloride ZnCI 2 = 136.3 (7646-85-7)
Clear, colourless, fiammable liquid, practically insoluble in Of the British Pharmacopoeia.
water, miscible with ethanol (96%). Zinc Chloride-Formic Acid Soludon Dissolve 20 g of
dig, about 0.881 ; ntO, about 1.505; boiling point, about zinc chloride in 80 g of an 85 % w/v solution of anhydrous
144°; melting point, about -25°. jormic acid.
Xylene Cyanol FF CI 42135; (2650-17-1) Zinc Chloride Solution, Iodinated Dissolve 20 g of zinc
A blue, alcohol-soluble dye used as a screening agent in chloride and 6.5 g of potassium iodide in 10.5 mL of water.
methyl orange-xylene cyanol FF solution. Add 0.5 g of iodine and shake for 15 minutes. Filter if
Xyleno10range [3H-2, 1-Benzoxathiol-3- necessary.
ylidenebis( 6-hydroxy-5-methyl-m- Store protected from light.
phenylene)methylenenitrilo] tetra-acetic acid S,S-dioxide Zinc Iodide Znl2 = 319.2 (10139-47-6)
tetrasodium salt, Tetrasodium 3,3'-(3H-2, l-benzoxathiol-3- General reagent grade of commerce.
ylidene)bis [( 6-hydroxy-5-methyl-3,1-
Zinc Iodide and Starch Solution To a solution of 2 g of
phenylene )methyleneiminobisacetate] S,S-dioxide;
zinc chlorzde in 10 mL of water add 0.4 g of soluble starch and
C31H2SN 2Na40 !3S = 761 (3618-43-7)
heat until the starch has dissolved. After cooling to room
Reddish-brown crystalline powder, soluble in water. temperature add 1 mL of a colourless solution containing
XylenoI Orange Soludon Shake 0.1 g of xylenol orange 0.10 g zinc as filings and 0.2 g of iodine in water, Dilute the
with 100 mL of water and filter, if necesssary. solution to 100 mL with water and filter. Store protected
Xylenol Orange Triturate from light.
Triturate 1 part of xylenol orange with 99 parts of potassium Sensitivity Dilute 0.05 mL of sodium nitrzú solution to
nitrate. 50 mL with water. To 5 mL of this solution add 0.1 mL of
Test/or sensitivity To 50 mL of water add 1 mL of dilute dilute sulfuric acid and 0.05 mL of the zinc iodide and starch
acenc acid, 50 mg of the xylenol orange triturate and solution and mix. The solution becomes blue.
0.05 mL of lead nitrate solution. Add hexamethylenetetramine Zinc Oxide ZnO = 81.4 (1341-13-2)
until the colour changes from yellow to violet-red. After Of the British Pharmacopoeia.
addition of 0.1 mL of 0.1 M sodium edetate the colour Zinc Powder Zn = 65.4 (7440-66-6)
changes to yellow.
Xylose D-Xylose; CSH100S = 150.1 (58-86-6)
Very fine, grey powder, soluble in dilute hydrochlorz'c acid.
Zinc Shot Zn = 65.38
D-Xylose See Xylose.
Zinc Zn = 65.4 (7440-66-6)
Shot, 0.5 to 2.0 mm (about 8 to 30 mesh).
Zinc Sulfate Zinc sulphate;
Silver-white cylinders, granules, pellets or filings with a blue ZnS04,7H 20 = 287.5 (7446-20-0)
sheen.
Zinc Sulfate Heptahydrate of the British Pharmacopoeia.
Arsenic (2.4.2, M ethod A) Maximum 0.2 ppm.
Zirconyl Chloride A basic salt corresponding
Dissolve 5. O g in a mixture of the 15 mL of hydrochloric acid approximately to the formula
and 25 mL of water prescribed. ZrOCI 2,8H 20 = 322.3 (15461-27-5)
Zinc Acetate Zinc acetate dihydrate, Content, minimum 96.0% of ZrCI 2 0,8H 2 0.
(C 2H 30 2h Zn,2H 20 = 219.5 (5970-45-6)
White or almost whiÍ:e, crystalline powder or crystals, freely
Bright white or almost white crystals, slightly effiorescent, soluble in water and in ethanol (96%).
freely soluble in water, soluble in ethanol (96%). It loses its
Assay Dissolve 0.600 g in a mixture of 5 mL of nitric acid
crystallisation water at 100°.
and 50 mL of water. Add 50.0 mL of 0.1 M silver nitrate and
dig, about 1.735; melting point, about 237°. 3 mL of dibutyl phthalate and shake. Using 2 mL of jerric
Zinc Acetate Soludon Mix 600 mL of water with ammonium sulfate solution R2 as indicator, titrate with 0.1 M
150 mL of glacial acetic acid, add 54.9 g of zinc aceta te and ammonium thiocyanate until a reddish-yellow colour is
stir to dissolve. Continue stirring while adding 150 mL of obtained.
concentrated ammonia. Cool to room temperature and adjust 1 mL of 0.1 M si/ver nitrate is equivalent to 16.11 mg of
with ammonia to pH 6.4. Dilute the mixture to ' 1 L with ZrCIzO,8H20.
water.
ZirconyI Nitrate A basic salt corresponding approximately
Zinc, Activated Place the zinc cylinders or pellets to be to the formula ZrO(N03)2,2H 20 (14985-18-3) .
activated in a conical fiask and add a sufficient quantity of a
A white or almost white powder or crystals, hygroscopic,
50 ppm solution of chloroplatinic acid to cover the metal.
soluble in water. The aqueous solution is a c1ear or at most
Allow the metal to remain in contact with the solution for
slightly opalescent Iiquid.
10 minues, wash, drain and dry immediately.
Arsenic (2.4.2, Method A) To 5 g of the activated zinc add
15 mL of hydrochloric acid, 25 mL of water, 0.1 mL of Zirconyl Nitrate Solution A 1 gIL solution in a mixture
stannous chloride solution and 5 mL of potassium iodide solution. of 40 mL of water and 60 mL of hydrochloric acid.
No stain is produced on the mercuric bromide papero
Activity Repeat the test for arsenic using the same reagents
and adding a solution containing 1 Jlg of arsenic.
An appreciable stain appears on the mercuric bromide papero
2014 Appendix 1 B V-A139
Recrystallise sulfanilic acid R from boiling water R . Filter and

B. Volumetric Reagents and Solutions dry 10 eonstant mass at 100-105 oc.
Terminology Zinc Zn = 65.4 (7440-66-6)
Volumetric solutions are prepared according to the usual Content: minimum 99.9 per cent.
chemical analytical methods. The accuracy of the apparatus
used is verified to ensure that it is appropriate for the Preparation and Standardisation
intended use. For each solution the preparation and standardisation of the
The concentration of volumetric solutions is indicated in most eommonly used strengths are described. Solutions more
terms of molar'ity (M) . The molarity of a solution is the concentrated than those described are prepared and
number of moles of substance contained in 1000 mL of the standardised using proportionate amounts of the reagents.
solution. A solution that contains x moles of substance per Aqueous solutions less concentrated than those deseribed are
litre is said to be XM. prepared by making an exact dilution of a more concentrated
solution with carbon dioxide-free water. The correction factors
Volumetric solutions do not differ from the prescribed
of these solutions are the same as those from whieh the
strength by more than 10% . The molarity ofthe volumetric
dilutions were prepared. Aqueous solutions of molarity below
solutions is determined by an appropriate number of
O.lM are freshly prepared using carbon dioxide-free water.
titrations. The repeatability does not exceed 0.2% (relative
standard deviation). The water used in preparing volumetric solutions complies
with the requirements ofthe monograph for Purified Water.
British Pharmacopoeia monographs In assays and
When used for the preparation of unstable solutions such as
other quantitative tests, solutions to be standardised before
potassium permanganate and sodium thiosulfate, it should be
use; inelude the letters VS after the name of the reagent.
freshly boiled and cooled. When a solution is to be used in
Primary Standards an assay in which the end point is determined by an
electrochemical process, the exact coneentration of the
The following materials, after drying under the specified solution must be determined in the same way.
conditions, are recommended for use as primary standards in The eomposition of the medium in which a volumetric
the standardisation of volumetric solutions. Analytical reagent solution is standardised should be the same as that in which
grade materials of commerce must be used. it is to be used.
Arsenic Trioxide Arsenious tri oxide All volumetric solutions should, if practicable, be prepared,
Sublime in a suitable apparatus and store over anhydrous standardised and used at 20 0 ; if a titration is carried out at a
silica gel. markedly different temperature from that at whieh the
Arsenious Trioxide AS 20 3 = 197.8 (1327-53-3) standardisation took place, a suitable temperature correction
Sublime arsenious trioxide R in a suitable apparatus. should be made.
Storage: over anhydrous silica gel R. Volumetric Solutions
Benzoic Acid C 7 H 60 2 = 122.1 (65-85-0)
Acetic Acid VS C2H 40 2 = 60.1
Sublime benzoic acid R in a suitable apparatus.
Dilute 6.0 g of glacial acetic acid R to 1000.0 mL with
Potassium Bromate KBr03 = 167.0 (7758-01-2) water R.
Recrystallise potassium bromate from boiling water, collect the Standardisation . To 25 .0 mL of acetic acid add 0.5 mL of
crystals and dry to constant weight at 180 0 • phenolphthalein solution R and titrate with 0.1 M sodium
Potassium Dichromate Dry to constant weight at 150 0 • hydroxide.
Potassium Hydrogen Phtha1ate C sH sK0 4 = 204.2 Ammonium Cerium(Iv) Nitrate VS Ammonium and
(877-24-7) eerium nitrate; (NH4)2Ce(N03)6 = 548.2
Recrystallise potassium hydrogen phthalate R from boiling For a O.lM solution Shake a solution containing 56 mL of
water R, collect the crystals at a temperature aboye 35 oC sulfuric acid and 54.82 g of ammonium cerium(IV) nitrate for
and dry to eonstant mass at 110 oC. 2 minutes and carefully add five successive 100-mL
Potassium Iodate Dry to eonstant weight at 130 0
• quantities of water, shaking after each addition. Dilute the
Sodium Carbonate, Anhydrous Sodium Carbonate. elear solution to 1000 mL with water.
Na2C03 = 106.0 (497-19-8) After 10 days, ascertain its exaet eoncentration in the
Filter at room temperature a saturated solution of sodium following manner. To 25 mL of the solution add 2 g of
carbonate R. Introduce slowly into the filtrate a stream of potassium iodide and 150 mL of water. Titrate immediately
carbon dioxide R with constant cooling and stirring. After with 0.1 M sodium thiosulfate VS, using 1 mL of starch solution
about 2 h, collect the precipitate on a sintered-glass filter as indicator. Each mL of O. lM sodium thiosulfate VS is
(2. 1.2) . Wash the filter with ieed water R containing carbon equivalent to 54.82 mg of (NH4)2Ce(N03k
dioxide. After drying at 100 oC to 105 oC, heat 10 eonstant Store protected from light.
mass at 270-300 oC, stirring from time to time. For a O.OlM solution To 100 mL of O.lM ammonium
Sodium Chloride NaCl = 58.44 (7647-14-5) cerium(IV) nitrate VS add, while cooling, 30 mL of sulfuric
To 1 volume of the saturated solution of sodium chloride R acid and sufficient water to produce 1000 mL.
add 2 volumes of hydrochloric acid R . Colleet the crystals Ammonium Cerium(Iv) Sulfate VS Ammonium
formed and wash with hydrochloric acid R1. Remove the Cerium(IV) Sulphate VS, Ammonium and eerium sulfate;
hydrochloric acid by heating on a water-bath and dry the 2(NH4)2S04,Ce(S04h,2H20 = 632.6
crystals to constant mass at 300 oC. For a O.lM solution Dissolve 65 g of ammonium ceriUm(lV)
Sulfanilic Acid Sulphanilic Aeid; C 6H 7 N0 3 S = 173.2 sulfate in a mixture of 30 mL of sulfuric acid and 500 mL of
(121-57-3) water. Allow to cool and dilute to 1000 mL with water.
V-A140 Appendix 1 B 2014
Ascertain its exact concentration in the following manner. alizarin S solution and titrate with the barium perchlorate
To 25 mL of the solution add 2 g of potassium iodide and solution until an orange-red colour appears. Each mL of
150 mL of water. Titrate immediately with 0.1 M sodium 0.05M sulfuric acid VS is equivalent to 16.81 mg of
thiosulfate VS using 1 mL of starch solution as indicator. Ba(CI0 4)2.
Each mL of O.IM sodium thiosulfate VS is equivalent to For a O.025M solution Dilute 500 mL of 0.05M barium
63.26 mg of (2NH4)2S04,Ce(S04)z,2H20. perchlorate VS to 1000 mL with buffer solution pH 3.7.
For a O.01M solution To 100 mL of O.IM ammonium Benzethonium Chloride VS, O.004M Dissolve 1.792 g of
cerium(IV) sulfate VS add, while cooling, 30 mL of sulfuric benzethonium chloride, previously dried to constant weight at
acid and sufficient water to produce 1000 mL. 100° to 105°, in sufficient water to produce 1000 mL.
Anunonium Iron(n) Sulfate VS Arnmonium Iron(n) Calculate the molarity of the solution from the content of
Sulphate, Ferrous Arnmonium Sulfate; (NH 4)zFe(S04)z, C27 H 42 ClN0 2 in the dried benzethonium chloride
6H 20 = 392.1 determined in the following manner. Dissolve 0.35 g of the
For a O.1M solution Dissolve 40 g of ammonium iron(IJ) dried substance in 30 mL of anhydrous acetic acid, add 6 mL
sulfate in 100 mL of 2M sulfuric acid and dilute with sufficient of mercury(IJ) acetate solution and carry out Method l for non-
freshly boiled and cooled water to produce 1000 mL. aqueous titration, Appendix VIII A, using 0.05 mL of crystal
Ascertain its exact concentration in the following manner. violet solution as indicator. Carry out a blank titration.
To 25 mL add 10 mL of 1M sulfuric acid and 1 mL of Each mL of 0.1 M perchloric acid VS is equivalent to 44.81 mg
orthophosphoric acid and titrate with 0.02M potassium of C 27 H 42 ClN0 2.
permanganate Vs. Each mL of 0.02M potassium permanganate O.OlM Bismuth Nitrate VS Dissolve 4.86 g of bismuth
VS is equivalent to 39.21 mg of (NH4hFe(S04)2,6H20. nitrate pentahydrate in 60 mL of dilute nitric acid and dilute to
Anunonium Iron(m) Sulfate VS Ammonium Iron(m) 1000.0 mL with water.
Sulphate VS, Ferric ammonium sulfate; Standardisation To 25.0 mL of the bismuth nitra te solution,
NH4Fe(S04h,12H 20 = 482.2 add 50 mL of water and titrate with 0.01 M sodium edetate
For a O.1M solution Dissolve 50 g of ammonium iron(m) using 0.05 mL of a 1 gIL solution of xylenol orange as
sulfate in a mixture of 300 mL of water and 6 mL of sulfuric indicator.
acid and dilute to 1000 mL with water. Bromine VS Bromide-bromate; Br2 = 159.8
Ascertain its exact concentration in the following manner. For a O.05M solution Dissolve 2.7835 g of potassium
To 25 mL add 3 mL of hydrochloric acid and 2 g of potassium broma te and 13 g of potassium bromide in sufficient water to
iodide, allow to stand for 10 minutes and titrate the liberated produce 1000 mL. (This solution is equivalent to 0.0167M
iodine with O.IM sodium thiosulfate VS using starch mucz1age as bromide-bromate of the European Pharmacopoeia).
indicator. Each mL of 0.1M sodium thiosulfate VS is equivalent Store in a dark amber-coloured bottle.
to 48.22 mg of NH4Fe(S04)2, 12H20.
Cerium(Iv) Sulfate VS Cerium(Iv) Sulphate VS, Cerium
Anunonium Thiocyanate VS NH4SCN = 76.12 sulfate; Ce(S04)2,4H 20 = 404.3
For a O.1M solution Dissolve 7.612 g in sufficient water to For a O.1M solution Dissolve 40.4 g of cerium(IV) sulfate in
produce 1000 mL. a mixture of 500 mL of water and 50 mL of sulfuric acid
Ascertain its exact concentration in the following manner. (96% w/w). Allow to cool and dilute to 1000 mL with water.
To 20 mL of O.IM sz1ver nitrate VS add 25 mL of water, Ascertain its exact concentration immediately before use in
2 mL of 2M nitric acid and 2 mL of ammonium iron(m) sulfate the following manner. To 20.0 mL of the cerium sulfate
solution R2 and titrate with the ammonium thiocyanate solution, add 1.6 g of potassium iodide, 100 mL of water and
solution until a reddish yellow colour is obtained. Each mL 40 mL of dz1ute sulfuric acid. Titrate irnmediately with O.lM
of 0.1M silver nitrate VS is equivalent to 7.612 mg of sodium thiosulfate VS using 0.8 mL of starch solution as
NH4SCN. indicator. Each mL of O.IM sodium thiosulfate VS is equivalent
Barium Chloride VS BaCI2,2H 20 = 244.3 to 40.43 mg of Ce(S04)z,4H 20.
For a O.1M solution Dissolve 24.4 g of barium chloride in Cetylpyridinium Chloride VS C21H3SCIN,H20 = 358.0
sufficient water to produce 1000 mL. For a O.005M solution Dissolve 1.8 g of cetylpyridinium
Ascertain its exact concentration in the following manner. chloride in 10 mL of ethanol (96%) and dilute to 1000 mL
To 10 mL of the solution add 60 mL of water, 3 mL of with water.
13.5M ammonia and 0.5 to 1 mg of phthalein purple as Ascertain its exact concentration in the following manner.
indicator and titrate with O.IM disodium edetate VS. As the Transfer 25 mL to a separating funnél, add 25 mL of
solution begins to decolorise, add 50 mL of ethanol (96%) chloroform, 10 mL of O.OlM sodium hydroxide and 10 mL of a
and titrate until the bluish violet colour is discharged. freshly prepared 0.5% w/v solution of potassium iodide. Shake
Each mL of 0.1 M disodium edetate VS is equivalent to well, allow to separate and discard the chloroform layer.
24.43 mg of BaCI2,2H 20. Shake the aqueous layer with three further 10-mL quantities
Barium Perchlorate VS Ba(Cl0 4 )2 = 336.2 of chloroform and discard the chloroform solutions.
For a O.05M solution Dissolve 15.8 g of barium hydroxide Add 40 mL of hydrochloric acid, cool and titrate with
in a mixture of 75 mL of water and 7.5 mL of perchloric acid, 0.005M potassium iodate VS until the solution becomes pale
adjust to pH 3 with perchloric acid and filter if necessary. brown in colour. Add 2 mL of chloroform and continue the
To the solution add 150 mL of ethanol (96%), dilute to titration, shaking vigorously and allowing the layers to
250 mL with water and add sufficient buffer solution pH 3.7 separate after each addition, until the chloroform becomes
to produce 1000 mL. colourless. Titrate a mixture of 20 mL of water, 10 mL of the
Ascertain its exact concentration immediately before use in potassium iodide solution and 40 mL of hydrochloric acid with
the following manner. To 5 mL of 0.05M sulfuric acid VS add 0.005M potassium iodate VS in the same manner.
5 mL of water, 50 mL o(acetate buffer pH 3. 7 and 0.5 mL of The difference between the titrations represents the amount
of potassium iodate required. Each mL of 0.005M potassium violet-pink colour changes to yellow. Each mL of
iodate VS is equivalent to 3.580 mg of C21H3SClN,H20 . 0.02M disodium edetate VS is equivalent to 1.308 mg of Zn.
Copper Sulfate VS Copper Sulphate VS; For a O.01M solution Dissolve 3.722 g of disodium edetate
CuS04,5H 20 = 249.7 in sufficient water to produce 1000 mL.
For a O.02M solution Dissolve 5.0 g of copper(Il) sulfate in Ascertain its exact concentration as described aboye but
water and dilute to 1000 mL with water. dilute 10 mL, in place of 25 mL, of this solution to 200 mL
Ascertain its exact concentration in the following manner. with water. Each mL of O.OlM disodium edetate VS is
To 20 mL add 2 g of sodium acetate and titrate with equivalent to 0.6538 mg of Zn.
0.02M disodium edetate VS, using 0.1 mL of pyridylazonaphthol Hydrochloric Acid VS HCl = 36.46
solution as indicator, until the colour changes from violet-blue For a O.1M solution Dilute 8.5 mL of hydrochloric acid
to bright green. Titrate slowly towards the end point. with sufficient water to produce 1000 mL.
Each mL of 0.02M disodium edetate VS is equivalent to Ascertain its exact concentration in the following manner.
4.994 mg of CuS04,5H 20. Dissolve 0.1 g of anhydrous sodium carbonate in 20 mL of
Cupriethylenediamine Hydroxide Solution Use a 1M water, add 0.1 mL of methyl orange solution and titrate with
solution in which the molar ratio of ethy1enediamine to the hydrochloric acid until the solution becomes reddish
copper is 2.00 ± 0.04. yellow. Boil for 2 minutes and continue the titration until the
Dioctyl Sodium Sulfosuccinate VS Dioctyl Sodium reddish yellow colour is restored. Each mL of
Sulphosuccinate VS; C2oH37Na07S = 444.6 O.lM hydrochloric acid VS is equivalent to 5.30 mg of
For a O.01M solution Dissolve 4.5 g of diocr:yl sodium Na2C03'
sulfosuccinate in warm water, cool and dilute to 1000 mL with For a 1M solution Dilute 103.0 g of hydrochloric acid to
water. 1000 mL with water.
Ascertain its exact concentration in the following manner. Ascertain its exact concentration as described for the O.IM
To 25 mL add 25 mL of a solution containing 20% w/v of solution using 1.000 g of anhydrous sodium carbonate dissolved
anhydrous sodium sulfate and 2% w/v of sodium carbonate, in 50 mL of water. Each mL of 1M hydrochloric acid VS is
50 mL of chloroform and 1.5 roL of bromophenol blue solution equivalent to 53.00 mg of Na2C03'
and mix. Titrate with O.OIM tetrabutylammonium iodide VS For a 2M solution Dilute 206.0 g of hydrochloric acid to
until about 1 mL from the end point. Stopper the flask, 1000 mL with water.
shake vigorously for 2 minutes and continue the titration, in For a 3M solution Dilute 309.0 g of hydrochloric acid to
0.05-mL increments, shaking vigorously and allowing the 1000 mL with water.
flask to stand for about 10 seconds after ea eh addition.
Continue the titration until a blue colour just appears in the For a 6M solution Dilute 618 .0 g of hydrochloric acid to
chloroform layer. Each mL of O.OlM tetrabur:ylammonium 1000 mL with water.
iodide VS is equivalent to 4.446 mg of C2oH37Na07S, lodine VS 12 = 253.8
Disodium Edetate VS Sodium edetate; For a O.5M solution Dissolve 127 g of iodine and 200 g of
ClQH14N2Na20S,2H20 = 372.2 potassium iodide in sufficient water to produce 1000 mL.
For a O.1M solution Dissolve 37.5 g of disodium edetate in Ascertain its exact concentration in the following manner.
sufficient water to produce 500 mL, add 100 mL of To 2 mL of the solution add 1 mL of 2M acetic acid and
1M sodium hydroxide and dilute to 1000 mL with water. 50 mL of water. Titrate with O.lM sodium thiosulfate VS using
Ascertain its exact concentration in the following manner. starch solution as indicator. Each mL of O.IM sodium thiosulfate
Dissolve 0.120 g of zinc, in granules, in 4 mL of VS is equivalent to 12.69 mg ofI.
7M hydrochloric acid and add 0.1 mL of bromine water. Boil to Store protected from light.
remove excess bromine, cool, add 2M sodium hydroxide until For a O.OSM solution Dissolve 20 g of potassium iodide in
the solution is weakly acidic or neutral and carry out the the minimum amount of water, add 12.7 g of iodine, allow to
method for the complexometric titration of zinc, dissolve and dilute to 1000 mL with water.
Appendix VIII D. Each mL of O.lM disodium edetate VS is Ascertain its exact concentration as described aboye but
equivalent to 6.54 mg of Zn. using 20 mL of the solution being examined and 30 mL of
Store in a polyethylene container. water.
For a O.05M solution Dissolve 18.6 g of disodium edetate in Store protected from light.
sufficient water to produce 1000 mL. For a O.01M solution Add 0.3 g of potassium iodide to
Ascertain its exact concentration as described below. Each 20 mL of 0.05M iodine VS and add sufficient water to
mL of 0.05M disodium edetate VS is equivalent to 3.269 mg of produce 100 mL.
Zn. Iron(n) Sulfate VS Iron(Il) Sulphate VS, Ferrous sulfate;
For a O.02M solution Dissolve 7.444 g of disodium edetate FeS04,7H 20 = 278.0
in sufficient water to produce 1000 mL. For a O.1M solution Dissolve 27.80 g of iron(ll) sulfate in
Ascertain i~s exact concentration in the following manner. 500 mL of 1M sulfuric acid and add sufficient water to
Dissolve 0.100 g of zinc, in granules, in 4 mL of produce 1000 mL.
7M hydrochloric acid and add 0.1 mL of bromine water. Boil to Ascertain its exact concentration irnmediately before use in
remove excess bromine, cool and dilute to 100 mL with the following manner. To 25 roL of the solution add 3 mL
water. Dilute 25 mL of this solution to 200 mL with water, of orthophosphoric acid and titrate immediately with
add about 50 mg of xylenol orange triturate and hexamine until 0.02M potassium permanganate VS. Each mL of
the solution becomes violet-pink and add 2 g of hexamine in 0.02M potassium permanganate VS is equivalent to 27.80 mg
excess. Titrate with the disodium edetate solution until the ofFeS04,7H20 .
Karl Fischer Reagent VS Iodosulfurous reagent second titration represents the amount of lithium methoxide
The apparatus, which must be kept c10sed and dry during required. Each mL of O.IM lithium methoxide VS is equivalent
the preparation, consists of a 3000- to 4000-mL round- to 12.21 mg of C7H60Z.
bottomed ftask with inlets for a thermometer and a stirrer Magnesium Chloride VS MgCI2,6H20 = 203.3
and fined with a drying tube. To 700 mL of anhydrous For a O.1M solution Dissolve 20.33 g of magnesium ehlonde
pyridine and 700 mL of 2-methoxyethanol add, stirring in sufficient water to produce 1000 mL.
constantly, 220 g of finely powdered iodine, previously dried Ascertain its exact concentration by carrying out the method
over phosphorus pentoxide. Continue stirring until the iodine for the complexometric titration of magnesium,
has completely dissolved (about 30 minutes), cool to -10° Appendix VIII D, using 25 mL of the magnesium chloride
and add quickly, while stirring, 190 g of sulfur dioxide. solution. Each mL ofO.lM disodium edetate VS is equivalent
Do not allow the temperature to exceed 30°; coo!. to 20.33 g of MgCI 2,6H20.
Determine the water-equivalent of the reagent immediately Magnesium Sulfate VS Magnesium Sulphate VS;
before use in the following manner. Transfer 20 mL of MgS0 4 ,7H zO = 246.5
anhydrous methanol to the titration vessel and titrate to the
For a O.05M solution Dissolve 12.5 g of magnesium sulfate
electrometric end point with the reagent, Appendix IX C.
in sufficient water to produce 1000 mL.
Add in an appropriate form a suitable amount of water,
accurately weighed, and titrate to the end point. Calculate Ascertain its exact concentration by carrying out the method
the water-equivalent of the reagent in mg per mL. for the complexometric titrau"on of magnesium,
The minimum water equivalent is 3.5 mg ofwater per mL of Appendix VIII D, using 40 mL of the magnesium sulfate
reagent. Work protected from humidity. solution. Each mL ofO.lM disodium edetate VS is equivalent
to 24.65 mg of MgS04 ,7H 20 .
The composiu"on of comrnercially available Karl Fischer reagents
ofien differs from that above by the replacement of pyridine with Mercury(n) Nitrate VS Mercuric nitra te; Hg(N0 3)2 + aq
other basic compounds. The. use of these reagents must be validated For a O.02M solution Dissolve 6.85 g of mercury(Il) nitrate
in ordér to verify in each individual case the stoichiometry and the in 20 mL of 1M nitric acid and add sufficient water to produce
absence of incompatibility between the substance under test and the 1000 mL.
reagent. Ascertain its exact concentration in the following manner.
O.IM Lanthanum Nitrate VS Dissolve 43.30 g of Dissolve 15 mg of sodium chlonde in 50 mL of water and
lanthanum nitra te R in water R and dilute to 1000.0 mL with titrate with the mercury nitrate solution determining the end
the same solvent. point potentiometrically, using a platinum or mercury
Standardisation. To 20 mL of the lanthanum nitra te solution, indicator electro de and a rnercury- mercury(l) sulfate
add 15 mL of water R and 25 mL of 0.1 M sodium edetate. reference electrode. Each mL of 0.02M mercury(Il) nitrate VS
Add about 50 mg of xylenol orange triturate R and about 2 g is equivalent to 2.338 mg of NaC!.
of hexamethylenetetramine R. Titrate with 0.1 M zinc sulfate Nitric Acid VS HN0 3 = 63.01
until the colour changes from yellow to violet-pink. For a 1M solution Dilute 96.6 g of nitric acid with
1 mL of 0.1 M sodium edetate is equivalent to 43.30 mg of sufficient water to produce 1000 mL.
La(N0 3 )3,6H zO. Ascertain its exact concentration in the following manner.
Lead Nitrate VS Pb(N0 3 )2 = 33l.2 Dissolve 2 g of anhydrous sodium carbonate in 50 mL of water
For a O.05M solution Dissolve 16.5 g of lead(lI) nitrate in and titrate with the nitric acid solution using 0.1 mL of
sufficient water to produce 1000 mL. methyl orange solution as indicator until the solution becomes
Ascertain its exact concentration in the following manner. reddish yellow. Boil for 2 minutes, cool and continue the
To 50 mL of the solution add 300 mL of water and carry out titration until the reddish yellow colour is restored. Each mL
the method for the complexometric titration of lead, of 1M nitric acid VS is equivalent to 53.00 mg ofNa2C03'
Appendix VIII D. Each mL of O.IM disodium edetate VS is Perchloric Acid VS HCI0 4 = 100.5
equivalent to 33.12 mg ofPbCN03)Z. For a O.1M solution To 900 mL of glacial aceu"c acid add
For a O.1M solution Dissolve 33.0 g of lead(Il) nitrate in 8.5 mL of perchloric acid, mix, add 30 mL of aceu"e anhydride,
sufficient water to produce 1000 mL. dilute to 1000 mL with glacial acetie acid, mix and allow to
Ascertain its exact concentration in the following manner. stand for 24 hours. Determine the water content,
To 20 mL of the solution add 300 mL of water and carry out Appendix IX C, without addition of methanol and if
the method for the complexometric titrau"on of lead, necessary adjust the water content to between 0.1 and 0.2%
Appendix VIII D . Each mL of disodiUll1 edetate VS is by adding either aceu"c anhydride or water, allow to stand for
equivalent to 33.12 mg of PbCN03h 24 hours.
Lithium Methoxide VS LiOCH 3 = 37.97 Ascertain its exact concentration in the following manner.
Dissolve 0.35 g of potassium hydrogen phthalate in 50 mL of
For a O.1M solution Dissolve in small. portions 0.694 g of anhydrous aeeu"c acid, warming gently if necessary. Allow to
lithium in 150 mL of anhydrous methanol and add sufficient cool protected from the air and titrate with the perchloric
toluene to produce 1000 mL. acid solution using 0.05 mL of crystal violet solution as
Ascertain its exact cóncentration immediately before use in indicator. Record the temperature at which the
the following manner. To 10 mL of dimethylformamide add standardisation was carried out. If the temperature at which
0.05 mL of a 0.3% w/v solurion of thymol blue in methanol an assay is carried out is different from that at which the
and titrate with the lithium methoxide solution until apure O.IM perchloric acid has been standardised, the volume used in
blue colour is produced. Immediately add 0.2 g of benzoic the assay becomes
acid, stir to effect solution and titrate with the lithium
Ve = V[l + O.OOII(tl - t2)]
methoxide solution until the pure blue colour is restored.
Protect the solution from atrnospheric carbon dioxide
throughout the titration. The volume of titrant used in the
where ti is the temperature during standardisation, t2 is the soludon as indica tor. Each mL of 0 .1M hydroehlon'e acid VS is
temperature during the assay, Vis the observed volume and equivalent to 5.611 mg ofKOH.
Ve is the corrected volume. For a O.SM solution Dissolve 3 g of potassium hydroxide in
Each mL of O.IM perchloric acid VS is equivalent to 20.42 mg 5 mL of water and add sufficient aldehyde-free ethanol (96%)
of Cs H sK0 4 . to produce 100 mL. Allow the solution to stand for 24 hours
For a O.02M solution Dilute 20.0 mL of O.IM perchloric and decant the c1ear solution.
acid to 100.0 mL with anhydrous acetic acid. Ascertain its exact concentration in the following manner.
Other strengths of perchloric acid should be prepared by Titrate 20 mL of the solution with O.5M hydroehlO1'ic acid VS
diluting O.IM perchloric acid VS appropriately with anhydrous using 0.5 mL of phenolphthalein solution as indicator.
acetic acid. Each mL of 0.5M hydroehloric acid VS is equivalent to
28.06 mg ofKOH.
Potassium Bromate VS KBr03 = 167 .0
Store in a well-c1osed container protected from Iight.
For a O.0083M solution Prepare by appropriate dilution of
a 0 .033M solution. Potassium Hydroxide in Ethanol (60%) VS Potassium
hydroxide in alcohol (60% v/v)
For a O.0167M solution Dissolve 2.783 g of potassiwn
bromate in sufficient water to produce 1000 mL. For a O.SM solution Prepare and standardise as directed
for O.5M ethanolic potassium hydroxide VS but using aldehyde-
For a O.02M solution Dissolve 3.340 g of potassium
free ethanol (60%) in place of the aldehyde-free ethanol
bromate in sufficient water to produce 1000 mL.
(96%).
For a O.033M solution Dissolve 5.567 g of potassiwn
Potassium Hydroxide in Ethanol (90%) VS
bromate in sufficient water to produce 1000 mL.
For a 1M solution Dissolve 60 g of potassiwn hydroxide in
Potassium Dichromate VS K2Cr207 = 294.2
sufficient aldehyde-free ethanol (90%) to produce 1000 mL,
For a O.0167M solution Dissolve 4.9 g of potassium allow the solution to stand for 24 hours, decant the c1ear
dichromate in sufficient water to produce 1000 mL. solution and ascertain its exact concentration by the method
Ascertain its exact concentration in the following manner. described under 0.5.\\ ethanolic potassium hydroxide VS.
To 20 mL of the solution add 1 g of potassium iodide and Potassium Iodate VS Kl0 3 = 214.0
7 mL of 2M hydrochloric acid. Add 250 mL of water and
For a O.OSM solution Dissolve 10.7 g of potassiU111 iodate in
titrate with O.IM sodium thiosulfate VS, using 3 mL of starch
solution as indicator, until the colour changes from blue to
Iight green. Each mL of O.IM sodium thiosulfate VS is Ascertain its exact concentration in the following manner.
equivalent to 4 .9 mg ofK2Cr207. Dilute 25 mL of the solution to i 00 mL with water and to
20 mL of this solution add 2 g of potassiw'/l iodide and 10 mL
Potassium Hydrogen Phthalate VS C SH S0 4 K = 204.2
of dilute sulfurie acid. Titrate with 0.1 M sodium thiosulfate VS
For a O.1M solution Dissolve 20.42 g of potassium hydrogen using 1 mL of stareh solution, added towards the end of the
phthalate in about 800 mL of anhydrous aceuc acid, heat on a titration, as indicator. Each mL of O. I M sodium thiosulfate VS
water bath until completely dissolved, protected from is equivalent to 3.566 mg of Kl0 3 .
humidity, cool to 20° and add sufficient anhydrous acetic acid
Potassium Iodide VS Kl = 166.0
to produce 1000 mL.
For a O.001M solution Dilute 10.0 mL of potassium iodide
Potassium Hydroxide VS KOH = 56.11
solution to 100.0 mL with water. Dilute 5 mL of this solution
For a O.1M solution Dissolve 6 g of potassium hydroxide in to 500.0 mL with water.
sufficient carbon dioxide-free water to produce 1000 mL.
Potassium Permanganate VS KMn0 4 = 158.0
Ascertain its exact concentration in the following manner.
For a O.02M solution Dissolve 3.2 g of potassium
Titrate 20 mL ofthe solution with O.IM hydroehlorie acid VS
permanganate in 1000 mL of water, heat on a water bath for
using 0.5 mL of phenolphthalein soludon as indicator.
1 hour, cool and filter through a sintered-glass filter.
Each mL of O.I M hydroehlon'c acid VS is equivalent to
5.611 mg ofKOH. Ascertain its exact concentration immediately before use in
the following manner. To 20 mL of the solution add 2 g of
For a 1M solution Dissolve 60 g of potassium hydroxide in
potassium iodide and 10 mL of 1M sulfuric acid and titrate with
sufficient earbon dioxide-free water to produce 1000 mL.
O. IM sodium thiosulfate VS using 1 mL of stareh solurion,
Ascertain its exact concentration in the following manner. added towards the end of the titration, as indicator. Each mL
Titrate 20 mL of the solution with 1M hydroehloric acid VS of O.IM sodium thiosulfate VS is equivalent to 3. 161 mg of
using 0.5 mL of phenolphthalein solution as indicator.
KMn04'
Each mL of 1M hydrochlorie acid VS is equivalent to
56.11 mg ofKOH.
Silver Nitrate VS AgN0 3 = 169.9
Potassium Hydroxide VS, Ethanolic Alcoholic
potassium hydroxide For a O.1M solution Dissolve l7.0 g of stlver nitrate in
For a O.01M solution Dilute 2.0 mL of 0.5M ethanolie
potassium hydroxide to 100.0 mL with aldehyde-free ethanol Ascertain its exact concentration in the following manner.
(96%). Dissolve 0.1 g of sodium ehloride in 30 mL of water and titrate
with the silver nitrate solution determining the end point
For a O.1M solution Dissolve 6 g of potassium hydroxide in
potentiometrically, Appendix VIII B. Each mL of O.IM silver
50 mL of water and dilute to 1000 mL with aldehyde-free
nitrate VS is equivalent to 5.844 mg ofNaCI.
ethanol (96%).
Ascertain its exact concentration immediately before use in
the following manner. Titrate 20 mL of the solution with For a O.001M solution Dilute 5 mL of O.IM silver nitrate
0.1 M hydrochloric acid VS using 0.5 mL of phenolphthalein VS to 500 mL with water.
Sodium Arsenite VS Ascertain its exact concentration immediately before use in

For a O.lM solution Dissolve 4.946 g of arsenic trioxide in the following manner. Dissolve 0.2 g of benzoic acid in a
a mixture of 20 mL of 10M sodium hydroxide and 20 mL of mixture of 10 mL of ethanol (96%) and 2 mL of water and
water, dilute to 400 mL with water and add 2M hydrochloric titrate with the ethanolic sodium hydroxide solution using
acid until the solution is neutral to litmus papel'. Dissolve 2 g 0.2 mL of thymolphthalein solution as indicator. Each mL of
of sodium hydrogen carbonate in the solution and dilute to O.lM ethanolic sodium hydroxide VS is equivalent to 12.21 mg
500 mL with water. of C 7 H 6 0 2 .
Sodium Dodecyl Sulfate VS Sodium Dodecyl Sulphate Sodium Methoxide VS
VSi C12H2SNa04S = 288.4 For a O.lM solution Cool 175 mL of anhydrous methanol
For a O.OOlM solution Dissolve 0.2884 g of sodium dodecyl in ice and add, in small portions, about 2.5 g of freshly cut
sulfate, ca1culated with reference to the substance dried at sodium. When the metal has dissolved add sufficient coluene
105 0 for 2 hours, in sufficient water to produce 1000 mL. to produce 1000 mL.
Ascertain its exact concentration in the following manner. Ascertain its exact concentration immediately before use in
To 50 mL add 15 mL of chloroform, 10 mL of 1M sulfuric the following manner. To 10 mL of dimethylformamide add
acid and 1 mL of a solution containing 0.003% w/v of each 0.05 mL of a 0.3 % w/v solution of thymol blue in methanol
of dimethyl yellow and oracet blue 2R in chloroform and titrate and titrate with the sodium methoxide solution until apure
with 0.004M benzethonium chloride VS, shaking vigorously and blue colour is produced. Immediately add 0.2 g of benzoic
allowing the layers to separate after each addition, until the acid, stir to effect solution and titrate with the sodium
chloroform layer acquires a permanent clear green colour. methoxide solution until the pure blue colour is restored.
Each mL of 0.004M benzethonium chloride VS is equivalent to Protect the solution from atrnospheric carbon dioxide
1.154 mg of C12H2SNa04S , throughout the titration. The volume of titrant used in the
second titration represents the amount of sodium methoxide
Sodium Hydroxide VS NaOH = 40.00
required. Each mL of O.IM sodium methoxide VS is equivalent
For a O.lM solution Dissolve 4.2 g of sodium hydroxide in to 12.21 mg of C 7 H 60 2.
sufficient carbon dioxide-free water to produce 1000 mL.
Sodium Nitrite VS NaN0 2 = 69.00
For a O.1M solution Dissolve 7.5 g of sodium nitrite in
the following manner. Titrate 20 mL of the solution with
O.IM hydrochloric acid VS using the indicator prescribed in the
assay in which the solution is to be used. Each mL of Ascertain its exact concentration immediately before use in
O.IM hydrochloric acid VS is equivalent to 4.00 mg ofNaOH. the following manner. Dissolve 0.3 g of sulfanilic acid in
50 mL of 2M hydrochloric acid, add 3 g of potassium bromide,
When a carbonate-free solution is specified it is prepared
cool in ice and titrate with O.IM sodium nitrite VS determining
using the following method. Dissolve sodium hydroxide in an
the end point electrometrically. Each mL of O.IM sodium
equal weight of water and allow to stand ovemight. Taking
nitrite VS is equivalent to 17.32 mg of C 6H 7 N0 3 S.
precautions to avoid absorption of carbon dioxide, siphon off
or decant the clear supematant liquid and dilute with carbon Sodium Periodate VS NaI0 4 = 213.9
dioxide-free water to the desired molarity. The solution For a O.lM solution Dissolve 21.4 g of sodium periodate in
complies with the following test. about 500 mL of water and add sufficient water to produce
Titrate 20 mL of hydrochloric acid VS of the same molarity as 1000 mL.
the solution being examined with the sodium hydroxide Ascertain its exact concentration in the following manner.
solution using 0.5 mL of phenolphthalein solution as indicator. To 20 mL of the solution in a stoppered fiask add 5 mL of
At the end point add just sufficient of the acid to discharge perchloric acid, close the fiask, shake, adjust the pH of the
the pink colour and boil to reduce the volume to 20 mL. solution to 6.4 using a saturated solution of sodium hydrogen
Add, whilst boiling, sufficient of the acid again to discharge carbonate, add 10 mL of porassium iodide solulÍon, close the
the pink colour which does not reappear after prolonged fiask, shake and allow to stand for 2 minutes. Titrate with
boiling. Not more than 0.1 mL of the acid is required. 0.025M sodium arsenite VS until the yellow colour almost
For a 1M solution Dissolve 42 g of sodium hydroxide in disappears, add 2 mL of starch solulÍon and titrate slowly until
sufficient carbon dioxide-free water to produce 1000 mL. the colour is completely discharged. Each mL of
0.025M sodium arsenite VS is equivalent to 5.345 mg of
the following manner. Titrate 20 mL of the solution with NaI0 4 ·
1M hydrochloric acid VS using the indicator prescribed in the Sodium Tetraphenylborate VS B(C6Hs)4Na = 342.2
assay in which the solution is to be used. Each mL of For a O.OlM solution Dissolve 3.5 g of sodium
1M hydrochloric acid VS is equivalent to 40.00 mg of NaOH. tetraphenylborate in 50 mL of water, shake for 20 minutes
When 0.1M sodium hydroxide VS is used in the assay of halide with 0.5 g of aluminium hydroxide gel, add 250 mL of water
salts of organic bases, ascertain its exact concentration in the and 16.6 g of sodium chloride and allow to stand for
following manner. Dissolve 0.100 g of benzoic acid in a 30 minutes. Filter, add 600 mL of water, adjust the pH to
mixture of 5 mL of O.OIM hydrochloric acid and 50 mL of 8.0 to 9.0 with O.I M sodium hydroxide and dilute to 1000 mL
ethanol (96%). Titrate with the solution being examined and with water.
note the volume added between the two points of infiection. Ascertain its exact concentration in the following manner.
Each mL of O. lM sodium hydroxide VS is equivalent to Dissolve 7 mg of potassium chloride, previously dried at 150 0
12.2 1 mg of C 7H 6 0. for 1 hour, in 5 mL of acera te buffer pH 3.7 and 5 mL of
Sodium Hydroxide VS, Ethanolic water, add 15 mL of the sodium tetraphenylborate solution,
allow to stand for 5 minutes and filter through a dry,
For a O.lM solution Add 3.3 g of 10M sodium hydroxide
sintered-glass filter. To 20 mL of the filtrate add 0.5 mL of
solulÍon to 250 mL of absolute echanol.
bromophenol blue solulÍon and titrate the excess of sodium
tetraphenylborate with 0.005M cetylpyridinium chlonde VS to
2014 Appendix 1 e V-A145
the blue colour of the indicator. Repeat the procedure O.IM tetrabutylammoniwn hydroxide VS is equivalent to
without the potassium chloride. The molarity of the solution 12.2 1 mg of C 7 H 60 2.
is given by the expression: Tetrabutylarnrnonium Hydroxide in Propan-2-o1 VS
aw/[15(a - b)0.07455] Tetrabutylammonium hydroxide in 2-propanol
where a is the volume of 0.005M cetylpyridinium chloride VS For a O.1M solution Prepare as described for
required when the potassium chloride is omitted, b is the O.IM tetrabutylammonium hydroxide VS using propan-2-o1 in
volume of 0.005M cetylpyridinium chloride VS required when place of toluene and ascertain its exact concentration
the potassium chloride is present and w is the weight, in g, of immediately before use as described aboye.
potassium chloride taken. Tetrabutylarnrnonium Iodide VS Cl6H36IN = 369.4
Sodiurn Thiosulfate VS Sodium Thiosulphate VS; For a O.01M solution Dissolve 4 g of tetrabutylammonium
Na2S20 3,5H20 = 248 .2 iodide in sufficient water to produce 1000 mL.
For a O.1M solution Dissolve 25 g of sodium thiosulfate and Ascertain its exact concentration in the following manner.
0.2 g of sodium carbonate in sufficient carbon dioxide-free water To 25 mL add 50 mL of O.OIM silver nitrate VS and 0.5 mL
to produce 1000 mL. of 2M nitric acid and titrate the excess of silver nitrate with
Ascertain its exact concentration in the following manner. O.OIM ammonium thiocyanate VS using ammonium iron(m)
To 20 mL ofO.0167Mpotassium bromate VS add 40 mL of sulfate solution RI as indicator. Each mL of O.OIM silver nitra te
water, 10 mL of potassium iodide solution and 5 mL of VS is equivalent to 3.694 mg of CI6H36IN.
7M hydrochloric acid. Titrate with the sodium thiosulfate Titaniurn(m) Chloride VS TiCI 3 = 154.3
solution using 1 mL of starch solution, added towards the end For a O.1M solution Dilute 100 mL of titaniwn(m) chlonde
of the titration, as indicator. Each mL of O.IM sodium solution with 200 mL of hydrochloric acid and add sufficient
thiosulfate VS is equivalent to 2.784 mg of KBr03' freshly boiled and cooled water to produce 1000 mL.
Sulfuric Acid VS Sulphuric Acid VS; H 2S0 4 = 98.08 Ascertain its exact concentration imrnediately before use by
For a O.5M solution Carefully add 28 mL of sulfuric acid titrating with it, in an atrnosphere of carbon dioxide, 25 mL
to 50 mL of water and dilute to 1000 mL with the same of O.IM ammonium iron(m) sulfate VS acidified with sulfuric
solvent. acid, using ammonium thiocyanate solution, added just before
Ascertain its exact concentration in the following manner. the end point, as indicator. Each mL of O.IM ammonium
Dissolve 1.0 g of anhydrous sodium carbonate in 50 mL of iron(m) sulfate VS is equivalent to 15.43 mg ofTiCl 3 .
water, add 0.1 mL of methyl orange solution and titrate with Zinc Chloride VS ZnCIz = 136.3
the sulfuric acid solution until the solution begins to tum For a O.05M solution Dissolve 6.82 g of zinc chloride,
reddish yellow. Boil the solution for 2 minutes, cool and weighed with appropriate precautions, in water. If necessary,
titrate again until the reddish yellow colour is restored. add 2M hydrochloric acid dropwise until the opalescence
Each mL of O.5M sulfun'c acid VS is equivalent to 53.00 mg disappears and add sufficient water to produce 1000 mL.
ofNa2C03'
Ascertain its exact concentration in the following manner.
For a O.05M solution Dilute 100 mL of O.5M sulfuric acid To 20 mL add 5 mL of 2M acetic acid and carry out the
VS to 1000 mL with water. method for the complexcmetric titration of zinc,
Ascertain its exact concentration as described aboye using Appendix VIII D . Each mL of O.I M disodium edetate VS is
0.10 g of anhydrous sodium carbonate dissolved in 20 mL of equivalent to 13.63 mg of ZnCI 2 .
water. Zinc Sulfate VS Zinc Sulphate VS; ZnS04,7H 20 = 287.5
Tetrabutylarnrnonium Hydroxide VS For a O.1M solution Dissolve 29 g of zinc sulfate in
C 16H 37NO = 259.5 sufficient water to produce 1000 mL.
For a O.1M solution Dissolve 40 g of tetrabutylammonium Ascertain its exact concentration in the following manner.
iodide in 90 mL of anhydrous methanol, add 20 g of finely To 20 mL add 5 mL of 2M acetic acid and carry out the
powdered silver oxide and shake vigorously for 1 hour. method for the complexometric titration of zinc,
Centrifuge a few mL of the mixture and test the supematant Appendix VIII D . Each mL of 0.1 M disodium edetate VS is
liquid for iodides. If a positive reaction is obtained, add a equivalent to 28.75 mg of ZnS04,7H 20 .
further 2 g of szlver oxide and shake for 30 minutes. Repeat
this procedure until the mixture is free from iodides, filter
through a fine sintered-glass filter and wash the reaction
vessel and filter with three 50-mL quantities of toluene.
Add the washings to the filtrate and add sufficient toluene to
C. Standard Solutions
produce 1000 mL. Pass dry carbon dioxide-free nitrogen The following solutions are used as reference standards in
through the solution for 5 minutes. limit tests and should, unless experience has shown it to be
unnecessary, be prepared imrnediately before use.
the following manner. To 10 mL of dimethylformamide add In monographs of the European Pharmacopoeia, the symbol
0.05 mL of a 0.3% w/v solution of thymol blue in methanol '%' may be replaced by the words 'per cent'. In such cases
and titrate with the tetrabutylarnrnonium hydroxide solution the reagent described below is to be used.
until apure blue colour is produced. Irnrnediately add 0.2 g Acetaldehyde Standard Solution (100 pprn C ZH 40)
of benzoic acid, stir to effect solution and titrate with the Dissolve 1.0 g of acetaldehyde R in 2-propanol R and dilute to
tetrabutylammonium hydroxide solution until the pure blue 100.0 mL with the same solvent. Dilute 5.0 mL of the
colour is resto red. Protect the solution from atrnospheric solution to 500.0 mL with 2-propanol R . Prepare immediately
carbon dioxide throughout the titration. The volume of before use .
titrant used in the second titration represents the amount of Acetaldehyde Standard Solution (100 pprn C 2 H 4 0) R1
tetrabutylammonium hydroxide required. Each mL of Dissolve 1.0 g of acetaldehyde R in water R and dilute to
100.0 mL with the same solvent. Dilute 5.0 mL of the
V-A146 Appendix 1 e 2014
solution to 500.0 mL with water R. Prepare irnmediately a solution in distilled water R containing bariUln chloride R
before use equivalent to 0.178 g ofBaCI 2,2H 20 in 100.0 mL.
Aluminium Standard Solution (200 ppm Al) Dissolve Barium Standard Solution (2 ppm Ba) Immediately
in water R a quantity of aluminiUln potassium sulfate R before use, dilute barium standard solution (50 ppm Ba) R to
equivalent to 0.352 g of AIK(S04)z,12H20. Add 10 mL of 25 times its volume with distilled water R.
dilute sulfuric acid R and dilute to 100.0 mL with water R. Bismuth Standard Solution (100 ppm Bi) Dissolve
Aluminium Standard Solution (100 ppm Al) bismuth R equivalent to 0.500 g of Bi in 50 mL of nitric
Irnmediately before use, dilute with water R to 10 times its acid R and dilute to 500.0 mL with water R. Dilute the
volume a solution containing 8.947 g of aluminium chloride R solution to 10 times its volume with dilute nitric acid R
in 1000.0 mL of water R. immediately before use.
Aluminium Standard Solution (10 ppm Al) Cadmium Standard Solution (0.1% Cd) Dissolve
Irnmediately before use, dilute with water R to 100 times its cadmium R equivalent to 0.100 g of Cd in the smallest
volume in a solution containing aluminium nitrate R necessary amount of a mixture of equal volumes of
equivalent to 1.39 g of AICN03)3,9H20 in 100.0 mL. hydrochloric acid R and water R and dilute to 100.0 mL with
Aluminium Standard Solution (2 ppm Al) Irnmediately a 1 per cent V/V solution of hydrochloric acid R.
before use, dilute with water R to 100 times its volume a Cadmium Standard Solution (10 ppm Cd) Immediately
solution containing aluminium potassium sulfate R equivalent before use, dilute cadmium standard solution (0.1 per cent
to 0.352 g of AIK(S04)z,12H 20 and 10 mL of dilute sulfuric Cd) R to 100 times its volume with a 1 per cent V/V
acid R in 100.0 mL. solution of hydrochloric acid R.
Ammonium Standard Soludon (100 ppm NH 4) Calcium Standard Soludon (1000 ppm Ca) Dissolve
Irnmediately before use, dilute to 25 mL with water R 2.5 g of calcium carbonate in 23 mL of 1M hydrochloric acid
10 mL of a solution containing ammonium chloride R and add sufficient distilled water to produce 100 mL. Dilute
equivalent to 0.741 g ofNH 4CI in 1000 mL. 1 volume of this solution to 10 volumes with distilled water
Ammonium Standard Solution (3 ppm NH4 ) irnmediately before use.
Irnmediately before use, dilute with water R to 100 times its Calcium Standard Solution (400 ppm Ca) Immediately
volume a solution containing ammonium chloride R equivalent before use, dilute with distilled water R to 10 times its volume
to 0.889 g ofNH4CI in 1000.0 mL. a solution in disulled water R containing calcium carbonate R
Ammonium Standard Solution (2.5 ppm NH4 ) equivalent to 1.000 g of CaC0 3 and 23 mL of 1 M
Irnmediately before use, dilute with water R to 100 times its hydrochloric acid in 100.0 mL.
volume a solution containing ammonium chlonde R equivalent Calcium Standard Solution (100 ppm Ca) Irnmediately
to 0.741 g of NH4CI in 1000.0 mL. before use, dilute with distilled water R to 10 times its volume
Ammonium Standard Solution (1 ppm NH4 ) a solution in distilled water R containing calcium carbonate R
Irnmediately before use, dilute ammonium standard solution equivalent to 0.624 g of CaC03 and 3 mL of acetic acid R in
(2.5 ppm NH,J R to 2.5 times its volume with water R . 250.0 roL.
Antimony Standard Solution (100 ppm Sb) Dissolve Calcium Standard Solution (100 ppm Ca), Alcoholic
antimony potassium tartrate R equivalent to 0.274 g of Irnmediately before use, dilute with ethanol (96 per cent) R to
C 4H 4K0 7 Sb,V2H 20 in 500 mL of 1M hydrochloric acid and 10 times its volume a solution in distilled water R containing
dilute the c1ear solution to 1000 mL with water R . calcium carbonate R equivalent to 2.50 g of CaC0 3 and
Antimony Standard Solution (1 ppm Sb) Dissolve 12 mL of acetic acid R in 1000.0 mL.
antimony potassium tamate R equivalent to 0.274 g of Calcium Standard Solution (100 ppm Ca) R1 Dilute
C 4H 4K0 7 Sb,1I2H20 in 20 mL of hydrochloric acid R1 and 1 volume of a solution of anhydrous calcium chloride
dilute the c1ear solution to 100.0 mL with water R. containing the equivalent of 0.2769% w/v of CaCIz in
To 10.0 mL ofthis solution add 200 mL of hydrochloric 2M hydrochloric acid to 10 volumes with water irnmediately
acid R1 and dilute to 1000.0 mL with water R. To 100.0 mL before use .
of this solution add 300 mL of hydrochloric acid R1 and Calcium Standard Soludon (10 ppm Ca) Irnmediately
dilute to 1000.0 mL with water R. Prepare the dilute before use, dilute with disulled water R to 100 times its
solutions irnmediately before use. volume a solution in distilled water R containing calcium
Arsenic Standard Solution (10 ppm As) Immediately carbonate R equivalent to 0.624 g of CaC0 3 and 3 mL of
before use, dilute with water R to 100 times its volume a acetic acid R in 250.0 roL.
solution prepared by dissolving arsenious trioxide R equivalent Chloride Standard Solution (50 ppm CI) Irnmediately
to 0.330 g of AS 20 3 in 5 mL of dilute sodium hydroxide before use, dilute with water R to 10 times its volume a
solution R and diluting to 250.0 mL with water R . solution containing sodium chloride R equivalent to 0.824 g of
Arsenic Standard Solution (1 ppm As) Irnmediately NaCI in 1000.0 mL.
before use, dilute arsenic standard solution (10 ppm As) R to Chloride Standard Solution (8 ppm CI) Immediately
10 times its volume with water R . before use, dilute with water R to 100 times its volume a
Arsenic Standard Solution (0.1 ppm As) Irnmediately solution containing sodium chlon·de R equivalent to 1.32 g of
before use, dilute arsenic standard solution (1 ppm As) R to NaCI in 1000.0 mL.
10 times its volume with water R . Chloride Standard Solution (5 ppm CI) Immediately
Barium Standard Solution (0.1% Ba) Dissolve barium before use, dilute with water R to 100 times its volume a
chloride R equivalent to 0.178 g of BaClz,2H 2 0 in distilled solution containing sodium chloride R equivalent to 0.824 g of
water R and dilute to 100.0 mL with the same solvent. NaCI in 1000.0 mL.
Barium Standard Solution (50 ppm Ba) Irnmediately Chromium Liposoluble Standard Solution (1000 ppm
before use, dilute with distilled water R to 20 times its volume Cr)l A chromium (metal) organic compound in an oil.
Chromium Standard Solution (0.1% Cr) Dissolve Glucose Standard Solution Dissolve 0.10 g of glucose in
potassiwn dichromate R equivalent ro 2.83 g of K2Cr20 7 in a saturated solution of benzoic acid in water, dilute ro
water R and dilute to 1000.0 mL with the same solvento 100 mL with the saturated benzoic acid solution and dilute
Chromium Standard Solution (100 ppm Cr) Dissolve 2 mL of this solution to 100 mL with water.
potassium dichromate R equivalent to 0.283 g of K2Cr207 in Glucose Standard Solution contains 20 ~lg of glucose
water R and dilute to 1000.0 mL with the same solvento per mL.
Chromium Standard Solution (0.1 ppm Cr) Glyoxal Standard Solution (20 ppm C 2 H 2 0 2) In a
Immediately before use, dilute chromium standard solution 100 mL graduated fiask weigh a quantity of glyoxal solution R
(lOO ppm Cr) R to 1000 times its volume with water R. corresponding ro 0.200 g of C 2H 2 0 2 and make up ro
Cobalt Standard Solution (100 ppm Co) Dissolve cobalt volume with anhydrous ethanol R. Immediately before use
nitrate R equivalent to 0.494 g of Co(NO)h,6H 2 0 in dilute the solution to 100 times its volume with the same
500 mL of 1M nitric acid and dilute the clear solution to solvento
1000 mL with water R. Glyoxal Standard Solution (2 ppm C 2H 2 0 2)
Copper Liposoluble Standard Solution (1000 ppm CU)l Irnmediately before use, dilute glyoxal standard solution
A copper (metal) organic compound in an oi!. (20 ppm C2H 2 0;J R ro 10 times its volume with anhydrous
Copper Standard Solution (0.1% Cu) Dissolve 0.393 g ethanol R.
of copper sulfate in sufficient water ro produce 100 mL. Hydrogen Peroxide Standard Solution (10 ppm H 2 0 2)
Copper Standard Solution (10 ppm Cu) Irnmediately Dilute 10.0 mL of dilute hydrogen peroxide solution R ro
300.0 mL with water R. Dilute 10.0 mL of this solution ro
before use, dilute copper standard solution (0.1 per cene Cu) R
to 100 times its volume with water R. 1000.0 mL with water R. Prepare immediately before use.
Iodide Standard Solution (20 ppm 1) Dilute 10.0 mL of
Copper Standard Solution (0.1 ppm Cu) Immediately
a 0.026% w/v solution of potassium iodide to 100.0 mL with
before use, dilute copper standard solution (la ppm Cu) R ro
water.
100 times its volume with water R.
Iodide Standard Solution (10 ppm 1) Irnmediately
Digitoxin Standard Solution Dissolve 0.1250 g of
digitoxin BPCRS in sufficient glacial acetic acid ro produce before use, dilute with water R ro 100 times its volume a
solution containing potassium iodide R equivalent ro 0.131 g
100.0 mL. Dilute 4.0 mL of the solution to 100.0 mL with
glacial acetic acid. To 25.0 mL of the resulting solution add of KI in 100.0 mL.
3.0 mL of water and mix wel!. Iron Standard Solution (0.1% Fe) Dissolve 0.100 g of
Fe in the smallest amount necessary of a mixture of equal
Elementary Standard Solutions for Atomic
Spectrometry, 1.000 gIL These solutions are prepared, volumes of hydrochloric acid R and water R and dilute to
generally in acidic conditions, from the element or salt of the 100.0 mL with water R.
element the minimum concentration of which is not less Iron Standard Solution (250 ppm Fe) Immediately
than 99.0%. The quantity per litre of solution is greater than before use, dilute with water R to 40 times its volume a
0.995 g throughout the guaranteed period, as long as the vial solution containing 4.840 g of jerric chlmide R in a 150 gil
has not been opened. The starting material (element or salt) solution of hydrochloric acid Rdiluted ro 100.0 mL.
and the characteristics of the final solvent (nature and Iron Standard Solution (20 ppm Fe) Immediately before
acidity, etc.) are mentioned on the labe!' use, dilute withwater R ro 10 times its volume a solution
Ferricyanide Standard Solution (50 ppm Fe(CN)6) containing jerric ammonium sulfate R equivalent to 0.863 g of
Immediately before use, dilute with water R to 100 times its FeNH4(S04)z,12H 2 0 and 25 mL of dilute sulfuric acid R in
volume a solution containing potassium jerricyanide R 500.0 mL.
equivalent ro 0.78 g ofK3Fe(CN)6 in 100.0 mL. Iron Standard Solution (10 ppm Fe) Immediately before
Ferrocyanide Standard Solution (100 ppm Fe(CN)6) use, dilute with water R to 100 times its volume a solution
Irnmediately before use, dilute with water R ro 10 times its containing jerrous ammonium sulfate R equivalent ro 7.022 g
volume a solution containing potassium jerrocyanide R of Fe(NH4)z(S04)z,6H 2 0 and 25 mL of dzlute sulfuric acid R
equivalent to 0.20 g of IZtFe(CN)6,3H 2 0 in 100.0 mL. in 1000.0 mL.
Fluoride Standard Solution (10 ppm F) Dissolve in Iron Standard Solution (8 ppm Fe) Irnmediately before
water R sodium fiuoride R previously dried at 300 oC for 12 h, use, dilute with water R to 10 times its volume a solution
equivalent to 0.442 g ofNaF, and dilute ro 1000.0 mL with containing 80 mg of iron R and 50 mL of hydrochloric acid R
the same solvent (1 mL = 0.2 mg F) . Store in a (220 gil HCl) in 1000.0 mL.
polyethylene container. Immediately before use, dilute the Iron Standard Solution (2 ppm Fe) Immediately before
solution ro 20 times its volume with water R. use, dilute iron standard solution (20 ppm Fe) R ro 10 times
Fluoride Standard Solution (1 ppm F) Immediately its volume with water R.
before use, dilute fiuoride standard solution (lO ppm F) R ro Iron Standard Solution (1 ppm Fe) Irnmediately before
10 times its volume with water R. use, dilute iron standard solution (20 ppm Fe) R ro 20 times
Formaldehyde Standard Solution (5 ppm CH2 0) its volume with water R.
Irnmediately before use, dilute with water R to 200 times its Lead Liposoluble Standard Solution (1000 ppm Pb)l A
volume a solution containing 1.0 g of CH 2 0 per litre lead (metal) organic compound in an oil.
prepared from jormaldehyde solution R. Lead Standard Solution (0.1% Pb) Dissolve lead
Germanium Standard Solution (100 ppm Ge) Dissolve nitrate R equivalent ro 0.400 g of Pb(N0 3)z in water R and
ammonium hexafiuorogermanate(IV) R equivalent to 0.307 g dilute to 250.0 mL with the same solvento
of (NH4)2GeF6 in a 0.01 per cent VIV solution of Lead Standard Solution(O.I% Pb) Rl Dissolve in dilute
hydrofiuoric acid R. Dilute the clear solution ro 1000 mL with lead-free nitric acid R a quantity of lead nitrate R equivalent to
water R . 0.400 g of Pb (N03)2 and dilute to 250.0 mL with the same
solvent.
Lead Standard Solution (100 ppm Pb) Immediately Mercury Standard Solution (1000 ppm Hg) Dissolve
before use, dilute lead standard solution (0.1 per cent Pb) R to mercuric chloride R equivalent ro 1.354 g of HgCl z in 50 mL
10 times its volume with water R . of dz1ute nitric acid R and dilute to 1000.0 mL with water R.
Lead Standard Solution (20 ppm Pb) Dissolve 0.80 g of Mercury Standard Solution (100 ppm Hg) Dissolve
lead(ll) nitrate in water containing 2 mL of nitric acid and add 1.080 g of yellow mercury (ll) oxide in the minimum volume of
sufficient water to produce 250 mL. Dilute 1 volume ro 2M hydrochloric acid and add sufficient water ro produce
100 volumes with water immediately before use. 1000 mL.
Lead Standard Solution (10 ppm Pb) Immediately Mercury Standard Solution (10 ppm Hg) Irnmediately
before use, dilute lead standard solution (100 ppm Pb) R ro before use, dilute with water to 100 times its volume a
10 times its volume with water R . solution containing mercuric chloride R equivalent to 0.338 g
Lead Standard Solution (10 ppm Pb) R1 Immediately of HgCl z in 250.0 mL.
before use, dilute with water R to 10 times its volume a Mercury Standard Solution (5 ppm Hg) Dilute 1.0 mL
solution containing 0.160 g of lead nitrate R in 100 mL of of a 0.0675% w/v solution of mercury(ll) chloride to 100.0 mL
water R, ro which is added 1 mL of lead-free nitric acid R and with water.
dilute ro 1000.0 mL. Nickel Liposoluble Standard Solution (1000 ppm Ni)!
Lead Standard Solution (10 ppm Pb) R2 Dilute lead A nickel (metal) organic compound in an oil.
standard solution (0. 1 per cent Pb) R1 to 100 times its volume Nickel Standard Solution (10 ppm Ni) Immediately
with dilute lead-free nitric acid R. Use within 1 week. before use, dilute with water R to 100 times its volume a
Lead Standard Solution (2 ppm Pb) Immediately before solution containing nickel sulfate R equivalent to 4.78 g of
use, dilute lead standard solution (10 ppm Pb) R to 5 times its NiS0 4 ,7HzO in 1000.0 mL.
volume with water R . Nickel Standard Solution (5 ppm Ni) Immediately
Lead Standard Solution (1 ppm Pb) Immediately before before use dilute nickel standard solution (10 ppm Ni) R ro
use, dilute lead standard solutwn (10 ppm Pb) R to 10 times twice its volume with water for chromatography R .
its volume with warer R. Nickel Standard Solution (0.2 ppm Ni) Immediately
Lead Standard Solution (0.5 ppm Pb) Dilute lead before use, dilute nickel standard solution (10 ppm Ni) R to
standard solution (10 ppm Pb) R2 to 20 times its volume with 50 times its volume with water R .
dz1ute lead-free nitric acid R . Use within 1 day. Nickel Standard Solution (0.1 ppm Ni) Irnmediately
Lead Standard Solution (0.25 ppm Pb) Immediately before use, dilute nickel standard solution (10 ppm Ni) R to
before use, dilute lead standard solution (1 ppm Pb) R ro 100 times its volume with water R.
4 times its volume with water R . Nitrate Standard Solution (100 ppm N0 3 ) Immediately
Lead Standard Solution (0.1 ppm Pb) Immediately before use, dilute with water R to 10 times its volume a
before use, dilute lead standard solution (1 ppm Pb) R ro solution containing potassium nitrate R equivalent to 0.815 g
10 times its volume with water R. of KN0 3 in 500.0 mL.
Lithium Standard Solution (100 ppm Li) Dissolve Nitrate Standard Solution (10 ppm N0 3 ) Irnmediately
0.6109 g of lithium chloride in sufficient water to produce before use, dilute nitrate standard solution (100 ppm NOJJ R
1000 mL. to 10 times its volume with water R.
Magnesium Standard Solution (0.1% Mg) Dissolve Nitrate Standard Solution (2 ppm N0 3) Immediately
magnesium sulfate R equivalent ro 1.010 g of MgS0 4 ,7H zO before use, dilute nitrate standard solution (10 ppm NOJJ R to
in distilled water R and dilute to 100.0 mL with the same 5 times its volume with water R .
solvento Nitrite Standard Solution (20 ppm NO z) Dissolve 0.6 g
Magnesium Standard Solution (1000 ppm Mg) of sodium nitrite in sufficient water to produce 100 mL and
Dissolve 5.275 g of magnesium nitrate R in 16 mL of dilure dilute 1 mL of this solution to 200 mL with water.
nitric acid R and dilute to 500.0 mL with water R.
Palladium Standard Solution (500 ppm Pd) Dissolve
Standardisation: carry out the determination of magnesium by 50.0 mg of palladium R in 9 mL of hydrochloric acid R and
complexometry (2.5. 11) . dilute to 100.0 mL with water R.
Magnesium Standard Solution (100 ppm Mg)
Palladium Standard Solution (20 ppm Pd) Dissolve
Immediately before use, dilute with water R to 10 times its
0.333 g of palladium chlO1"ide R in 2 mL of warm hydrochloric
volume a solution containing magnesium sulfate R equivalent
acid R. Dilute the solution to 1000.0 mL with a mixture of
ro 1.010 g of MgS0 4 ,7H zO in 100.0 mL.
equal volumes of dz1ute hydrochloric acid R and water R .
Magnesium Standard Solution (10 ppm Mg) Immediately befo re use dilute to 10 times its volume with
Immediately before use, dilute magnesium standard solution water R.
(100 ppm Mg) R to 10 times its volume with water R.
Palladium Standard Solution (0.5 ppm Pd) Dilute
Magnesium Standard Solution (10 ppm Mg) R1
1 mL of palladium standard solutwn (500 ppm Pd) R to
Immediately before use, dilute with water R to 100 times its
1000 mL with a mixture of 0.3 volumes of nitric acid R and
volume a solution containing 8.365 g of magnesium chloride R 99.7 volumes of water R.
in 1000.0 mL of dilute hydrochloric acid R.
Phosphate Standard Solution (200 ppm P0 4) Dissolve
Manganese Standard Solution (1000 ppm Mn) Dissolve
potassium dihydrogen phosphate R equivalent to 0.286 g of
manganese sulfate R equivalent to 3.08 g of MnS04,H zO in
KHzP04 in water R and dilute to 1000.0 mL with the same
500 mL of 1 M nitric acid and dilute the solution to
solvento
1000 mL with water R.
Phosphate Standard Solution (100 ppm P0 4 ) Dilute
Manganese Standard Solution (100 ppm Mn) Dissolve
10.0 mL of a 0.143 % w/v solution of potassium dihydrogen
manganese sulfate R equivalent to 0.308 g of MnS04,H zO in
orthophosphate to 100.0 mL with water immediately before
500 mL of 1M nitric acid and dilute the clear solution to
1000 mL with water R. use.
Phosphate Standard Solution (5 ppm P0 4) dipotassium sulfate R equivalent to 0.181 g of K 2S0 4 in

Irnrnediately before use, dilute with water R to 100 times its 100.0 mL.
volume a solution containing potassium dihydrogen Sulfate Standard Solution (10 ppm S04) Rl Sulphate
phosphate R equivalent to 0.716 g of KH 2P0 4 in 1000.0 mL. Standard Solution (lO ppm S04) Rl
Platinum Standard Solution (30 ppm Pt) Immediately Immediately before use, dilute with ethanol (30 per cent
before use, dilute with 1 M hydrochloric acid to 10 times its V/r-? R to 100 times its volume a solution containing
volume a solution containing 80 mg of chloroplatinic acid R in dipotassium sulfate R equivalent to 0.181 g ofK2S0 4 in
100.O mL of 1 M hydrochloric acid. 100.0 mL of ethanol (30 per eent V/ r-? R .
Potassium Standard Solution (0.2 % K) Dissolve Sulfite Standard Solution (80 ppm S02) Sulphite
dipotassium sulfate R equivalent to 0.446 g of K 2S0 4 in Standard Solution (80 ppm S02)
distilled water R and dilute to 100.0 mL with the same Dissolve 3.150 g of anhydrous sodium suljite R in freshly
solvent. prepared distilled water R and dilute to 100.0 mL with the
Potassium Standard Solution (600 ppm K) Irnrnediately same solvento Dilute 0.5 mL to 100 .0 mL with freshly
before use, dilute with water R to 20 times its volume a prepared distilled water R.
solution containing dipotassium sulfate R equivalent to 2.676 g Sulfite Standard Solution (1.5 ppm S02) Sulphite
ofK2S0 4 in 100.0 mL. Standard Solution (1.5 ppm S02)
Potassium Standard Solution (100 ppm K) Irnrnediately Dissolve sodium metabisuljite R equivalent to 0.152 g of
before use, dilute with water R to 20 times its volume a Na2S20S in water R and dilute to 100.0 mL with the same
solution containing dipotassium sulfate R equivalent to 0.446 g solvento Dilute 5.0 mL ofthis solution to 100.0 mL with
of K 2S0 4 in 100.0 mL. water R. To 3.0 mL of the resulting solution, add 4.0 mL of
Potassium Standard Solution (20 ppm K) Immediately 0.1 M sodium hydroxide and dilute to 100.0 mL with water R.
before use, dilute potassium standard solution (100 ppm K) R Thallium Standard Solution (10 ppm TI) Dissolve
to 5 times its volume with water R. thallous sulfate R equivalent to 0.1235 g ofTl2S0 4 in a 9 giL
Selenium Standard Solution (100 ppm Se) Dissolve solution of sodium chloride R and dilute to 1000.0 mL with
0.100 g of selenium R in 2 mL of nitrie acid R. Evaporate to the same solution. Dilute 10.0 mL of the solution to
dryness. Take up the residue in 2 mL of water R and 100.0 mL with the 9 gIL solution of sodium chloride R .
evaporate to dryness; carry out three times. Dissolve the Tin Liposoluble Standard Solution (1000 ppm Sn) A
residue in 50 mL of dilute hydroehloric acid R and dilute to tin (metal) organic compound in an Qi!.
1000.0 mL with the same acid.
Tin Standard Solution (5 ppm Sn) Dissolve fin R
Selenium Standard Solution (1 ppm Se) Immediately equivalent to 0.500 g of Sn in a mixture of 5 mL of water R
before use, dilute with water R to 40 times its volume a and 25 mL of hydrochloric acid R and dilute to 1000.0 mL
solution containing selenious acid R equivalent to 6.54 mg of with water R. Dilute the solution to 100 times its volume
H 2Se0 3 in 100.0 mL. with a 2.5 per cent V/V solution of hydrochloric acid R
Silver Standard Solution (5 ppm Ag) Irnrnediately irnrnediately before use.
before use, dilute with water R to 100 times its volume a Tin Standard Solution (0.1 ppm Sn) Immediately before
solution containing s¡Jver nitrate R equivalent to 0.790 g of use, dilute tin standard solution (5 ppm Sn) R to 50 times its
AgN0 3 in 1000.0 mL. volume with water R.
Sodium Standard Solution (1000 ppm Na) Dissolve a Titanium Standard Solution (100 ppm Ti) Dissolve
quantity of anhydrous sodium carbonate R equivalent to 100.0 mg of titanium R in 100 mL of hydroehloric acid R
2.305 g of Na ZC0 3 in a mixture of 25 mL of water R and diluted to 150 mL with water R, heating if necessary. Allow
25 mL of nitric acid R and dilute to 1000.0 mL with water R . to cool and dilute to 1000 mL with water R .
Sodium Standard Solution (200 ppm Na) Immediately Vanadium Standard Solution (1 gIL V) Dissolve in
before use, dilute with water R to 10 times its volume a water R ammonium vanadate R equivalent to 0.230 g of
solution containing sodium eh/oride R equivalent to 0.509 g of NH4 V0 3 and dilute to 100.0 mL with the same solvento
NaCl in 100.0 mL.
Water, Standard Solution for the Micro Deternúnation
Sodium Standard Solution (50 ppm Na) Dilute the of Standard solution of commerce for the coulometric
sodium standard solution (200 ppm Na) R to four times its determination of water containing a certified content of
volume with water R. water in a suitable solvento
Strontium Standard Solution (1.0 per cent Sr) Cover Zinc Standard Solution (5 mg/mL Zn) Dissolve 3.15 g
with water R, strontium carbonate R equivalent to 1.6849 g of of zinc oxide R in 15 mL of hydrochloric acid R and dilute to
SrC0 3 . Cautiously add hydrochloric acid R until all the solid 500.0 mL with water R .
has dissolved and there is no sign of further effervescence.
Zinc Standard Solution (100 ppm Zn) Irnrnediately
Dilute to 100.0 mL with water R .
before use, dilute with water R to 10 times its volume a
Sulfate Standard Solution (100 p~m S04) Sulphate solution containing zinc sulfate R equivalent to 0.440 g of
Standard Solution (100 ppm S04) ZnS04,7H 20 and 1 mL of aeetie acid R in 100.0 mL.
Immediately before use, dilute with distilled water R to Zinc Standard Solution (25 ppm Zn) Dilute 25.0 mL of
10 times its volume a solution in distilled water R containing zinc standard solution (100 ppm Zn) to 100.0 mL with water
dipotassium sulfate R equivalent to 0.181 g ofK2S0 4 in immediately before use.
100.0 mL.
Zinc Standard Solution (10 ppm Zn) Irnrnediately
Sulfate Standard Solution (10 ppm S04) Sulphate before use, dilute zinc standard so/ution (100 ppm Zn) R to
Standard Solution (10 ppm S04) 10 times its volume with water R.
Immediately before use, dilute with distilled water R to
100 times its volume a solution in distilled water R containing
2014
V-A150 Appendix 1 D
Zinc Standard Solution (5 ppm Zn) Immediately b~fore extract is colourless. Filter the aqueous layer to remove
use, dilute zinc standard solulion (lOO ppm Zn) R ro 20 tImes traces of carbon tetrachloride.
its volume with water R . Acetate Buffer Solution pH 4.7 RI Dissolve 136.1 g of
Zirconium Standard Solution (1 gIL Zr) Dissolve sodium acetate R in 500 mL of water R. Mi.." 250 mL of this
zirconyl nitrate R equivalent to 0.293 g of ZrO(N03)2,2H20 solution with 250 mL of dilute acetic acid R.
in a mixture of 2 volumes of hydrochloric acid R and Acetate Buffer Solution pH 5.0 To 120 mL of a 6 gIL
8 volumes of water R and dilute to 100.0 mL with the same solution of glacial acetic acid R add 100 mL of 0.1 M
mixture of solvents. potassium hydroxide and about 250 mL of water R. Mix.
Adjust the pH to 5.0 with a 6 giL solution of acetic acid R or
with 0.1 M potassium hydroxide and dilute ro 1000.0 mL with
water R.
D. Buffer Solutions Acetate Buffer Solution pH 6.0 Dissolve 100 g of
Buffer solutions should be prepared using carbon dioxide-free armnonium acetate R in 300 mL of water R, add 4.1 mL of
water. glacial acetic acid R, adjust the pH if necessary using
Acetate Buffer pH 2.45 Mix 200 mL of 1M hydrochloric ammonia R or acetic acid R and dilute ro 500.0 mL with
acid with 200 mL of 1M sodium aceta te and dilute to 1000 mL water R.
with water. Immediately before use adjust the pH ro 2.45 by Acetate-edetate Buffer Solution pH 5.5 Dissolve 250 g
the addition of 1M hydrochloric acid or 1M sodium aceta te, as of ammonium acetate R and 15 g sodium edetate R in 400 mL
required. of water R and add 125 mL of glacial acetic acid R.
Acetate Buffer pH 2.8 Dissolve 4 g of anhydrous sodium Acetone Solution, Buffered Dissolve 8.15 g of sodium
acetate in about 840 mL of water, add sufficient glacial acetic aceta te R and 42 g of sodium chloride R in water R, add
acid to adjust the pH to 2.8 (about 155 mL) and dilute to 68 mL of 0.1 M hydrochloric acid and 150 mL of acetone R
1000 mL with water. and dilute ro 500 mL with water R .
Acetate Buffer pH 3.4 Mix 5 volumes of O.lM sodium Arnmonia Buffer pH 10.0 Ammonium chloride buffer
acetate with 95 volurnes of O.lM acetic acid. solution pH 10.0
Acetate Buffer pH 3.5 Buffer solution pH 3.5 Dissolve 5.4 g of ammonium chloride R in 20 mL of water R,
Dissolve 25 g of ammonium acetate in 25 mL of water and add .35.0 mL of ammonia R and dilute ro 100.0 mL with
add 38 mL of 7M hydrochloric acid. Adjust the pH to 3.5 with water R .
either 2M hydrochloric acid or 6M ammonia and dilute to Arnmonia Buffer pH 10.9 Buffer solution pH 10.9
100 mL with water. Dissolve 6.75 g of ammonium chloride R in ammonia R and
Acetate Buffer pH 3.7 Dissolve 10 g of anhydrous sodium dilute to 100.0 mL with the same solvento
acetate in 300 mL of water, adjust ro pH 3.7 with glacial Arnmonia Buffer pH 10.9, Dilute Dilute 2 mL of
acetic acid and dilute to 1000 mL with water. If necessary, ammonia buffer pH 10.9 ro 1000 mL with water.
readjust ro pH 3.7 with glacial acetic acid or anhydrous sodiwn Arnmonium Carbonate Buffer Solution pH 10.3, O.IM
acetate as required, before use. Dissolve 7.91 g of ammonium carbonate R in 800 mL of
Acetate Buffer pH 404 Acetate buffer solution pH 4.4 water R. Adjust the pH (2.2.3) with dilute sodium hydroxide
Dissolve 136 g of sodium acetate R and 77 g of ammonium solution R. Dilute to 1000.0 mL with water R .
acetate R in water R and dilute to 1000.0 mL with the same Arnmonium Chloride Buffer Solution pH 9.5
solventó add 250.0 mL of glacial acetic acid R and mix. Ammonia buffer pH 9.5
Acetate Buffer pH 4.6 Acetate buffer solution pH 4.6 Dissolve 33.5 g of ammonium chloride R in 150 mL of
Dissolve 5.4 g of sodium acetate R in 50 mL of water R , add water R, add 42.0 mL of concentrated ammonia R and dilute
2.4 g of glacial acetic acid R and dilute to 100.0 mL with ro 250.0 mL with water R .
water R. Adjust the pH (2.2.3) if necessary. Storage: in a polyethylene container.
Acetate Buffer pH 5.0 Dissolve 13.6 g of sodium acetate Arnmonium Chloride Buffer Solution pH 10.0 Dissolve
and 6 mL of glacial acetic acid in sufficient water ro produce 5.4 g of ammonium chlO/ide R in 20 mL of water R, add
1000 mL. 35.0 mL of ammonia R and dilute ro 100.0 mL with
water R .
Acetate Buffer pH 6.0 Acetate buffer solution pH 6.0
Arnmonium Chloride Buffer Solution pH lOA Dissolve
Dissolve 100 g of ammonium acetate R in 300 mL of water R,
70 g of ammonium chloride R in 200 mL of water R, add
add 4.1 mL of glacial acetic acid R, adjust the pH (2.2.3) if
330 mL of concentrated ammonia R and dilute ro 1000.0 mL
necessary using ammonia R or acetic acid R and dilute ro with water R. If necessary, adjust to pH 10.4 with
500.0 mL with water R.
ammonia R.
Acetate Buffer Solution pH 4.4 Dissolve 136 g of sodium Arnmonium Chloride Buffer Solution pH 10.7 Dissolve
aceta te R and 77 g of ammoniwn acetate R in water R and 67.5 g of ammonium chloride R in water R, add 570 mL of
dilute to 1000.0 mL with the same solventó add 250.0 mL of concentrated ammonia R and dilute to 1000.0 mL with
glacial acetic acid R and mix. water R.
Acetate Buffer Solution pH 4.5 Dissolve 77 .1 g of Barbital Buffer Solution pH 7 A Mix 50 mL of a
ammonium acetate R in water R. Add 70 mL of glacial acetic solution in water R containing 19.44 gIL of sodiwn acetate R
acid R and dilute to 1000.0 mL with water R. and 29.46 giL of barbital sodiwn R with 50.5 mL of 0.1 M
Acetate Buffer Solution pH 4.7 Dissolve 136.1 g of hydrochloric acid, add 20 mL of an 85 gIL of sodium
sodium acetate R in 500 mL of water R. Mix 250 mL of this chloride R and dilute ro 250 mL with water R .
solution with 250 mL of dilute acetic acid R. Shake twice with Barbital Buffer Solution pH 8.4 Dissolve 8.25 g of
a freshly prepared, filtered, 0.1 giL solution of dithizone R in barbital sodiwn R in water R and dilute ro 1000.0 mL with
chlorofoml R . Shake with carbon tetrachloride R until the the same solvento
2014 AppendixID V-A151
Barbital Buffer Solution pH 8.6 R1 Dissolve in water R Buffer (Acetate) Solution pH 5.0 To 120 rnL of a
1.38 g of barbital R, 8.76 g of barbiral sodium R and 0.38 g of 0.6% w/v solution of glacial acetic acid add 100 rnL of
ealciwl1 lactate R and dilute to 1000.0 rnL with the sarne O.IM potassium hydroxide and about 250 rnL of water and
solvent. rnix. Adjust the pH to 5.0 with a 0.6 % w/v solution of glacial
Barbitone Buffer pH 7.4 Barbital buffer solution pH 7.4 acetic acid or with O.IMpotassium hydroxide and dilute to
Mix 50 rnL of a solution in water R containing 19.44 gIL of 1000 rnL with water.
sodium acetate R and 29.46 gIL of barbital sodium R with Buffer (HEPES) Solution pH 7.5 Dissolve 2.38 g of
50.5 rnL of 0.1 M hydroehlorie acid, add 20 rnL of an 85 gIL 2-[4- (2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid R in
of sodium ehlmide R and dilute to 250 rnL with water R. about 90 rnL of water R. Adjust the pH to 7.5 with sodium
hydroxide solution R. Dilute to 100 rnL with water R .
Barbitone Buffer pH 8.4 Barbital buffer solution pH 8.4
Dissolve 8.25 g of barbital sodium R in water R and dilute to Buffer (Phosphate) Solution pH 9.0 Dissolve 1.74 g of
1000.0 rnL with the sarne solvento potassium dihydrogen phosphate R in 80 rnL of water R , adjust
the pH (2. 2.3) with 1 M potassium hydroxide and dilute to
Barbitone Buffer pH 8.6 R1 Barbital buffer solution
100.0 rnL with water R .
pH 8.6 Rl
Buffer Solution pH 2.0 Dissolve 6.57 g of potassium
Dissolve in water R 1.38 g of barbital R, 8.76 g of barbiral
ehlon'de R in water R and add 119. O rnL of O. 1 M hydroehlorie
sodiwl1 R and 0.38 g of calcium lacta te R and dilute to
acid. Dilute to 1000.0 rnL with water R .
1000.0 rnL with the sarne solvento
Buffer Solution pH 2.2 Dissolve 100 g of porassium
Borate Buffer pH 7.5 Borate buffer solution pH 7.5
dihydrogen phosphate R in 800 rnL of water R; adjust to
Dissolve 2.5 g of sodiwl1 ehloride R, 2.85 g of disodium pH 2.5 (2.2.3) with hydroehlon'e acid R and dilute to
tetraborate R and 10.5 g of boric acid R in water R and dilute 1000.0 rnL with water R.
to 1000.0 rnL with the sarne solvento Adjust the pH (2.2.3) if
Buffer Solution pH 2.5 Dissolve 100 g of porassium
necessary.
dihydrogen onhophosphate in 800 rnL of water, adjust to
Swrage: at 2 oC to 8 cc. pH 2.5 with hydroehloric acid and add sufficient water to
Borate Buffer pH 8.0 To 50 rnL of a solution containing produce 1000 rnL.
0.6189 g of bmic acid and 0.7456 g of potassium chloride add Buffer Solution pH 2.5 R1 To 4.9 g of dilure phosphon'c
3.97 rnL of 0.2M sodium hydroxide VS and dilute to 200 rnL acid R add 250 rnL of water R. Adjust the pH (2.2.3) with
with water. dz7ute sodium hydroxide solurion R and dilute to 500.0 rnL with
At 20°, the solution rnay be used as a solution of standard water R.
pH. Buffer Solution pH 3.0 Dissolve 21.0 g of citric acid R in
Borate Buffer pH 8.4, 0.2M Dissolve 2.0 g of sodium 200 mL of 1 M sodium hydroxide and dilute to 1000 rnL with
hydroxide and 12. 1 g of bOlic acid in 250 rnL of water, adjust water R. Dilute 40. 3 rnL of this solution to 100 .0 rnL with
to pH 8.4, if necessary, by adding a few granules of boric 0. 1 M hydrochloric acid.
acid. Buffer Solution pH 3.5 Dissolve 25.0 g of ammonium
Borate Buffer pH 9.0 Buffer solution pH 9.0 aeerate R in 25 rnL of water R and add 38.0 rnL of
Dissolve 6.18 g of bOlic acid R in 0.1 M potassium chloride R hydroehlorie acid R1. Adjust the pH (2.2. 3) if necessary with
and dilute to 1000.0 rnL with the sarne solvento dilute hydrochlmic acid R or dilure ammonia R1. Dilute to
Mix 1000.0 rnL ofthis solution and 420 .0 rnL ofO.l M 100.0 rnL with water R.
sodium hydroxide. Buffer Solution pH 3.6 To 250.0 rnL of 0.2 M potassium
Borate Buffer pH 9.6 To 50 rnL of a solution containing hydrogen phthalate R add 11.94 mL of 0.2 M hydrochlorie
0.6189 g of boric aeid and 0.7456 g of potassium chlonde add acid. Dilute to 1000.0 rnL with water R.
36.85 rnL of 0.2M sodium hydroxide VS and dilute with water Buffer Solution pH 3.7 Ethanolic acetic-arnrnonia buffer
to 200 rnL. pH 3.7.
At 20°, the solution rnay be used as a solution of standard To 15.0 rnL of acetie acid R add 60 rnL of ethanol
pH. (96 per eent) R and 20 rnL of water R. Adjust to pH 3.7
Borate Buffer Solution pH 7.5 Dissolve 2.5 g of sodium (2.2.3) by the addition of ammonia R . Dilute to 100.0 rnL
ehlonde R, 2.85 g of disodium tetraborate R and 10.5 g of boric with water R .
acid R in water R and dilute to 1000.0 mL with the sarne Buffer Solution pH 5.2 Dissolve 1.02 g of potassium
solvento Adjust the pH if necessary. hydrogen phthalate R in 30.0 rnL of 0.1 M sodium hydroxide.
Swrage: at 2 cC to 8 oc. Dilute to 100.0 rnL with water R.
Borate Buffer Solution pH 8.0, 0.0015M Dissolve At 20°, the solution rnay be used as a solution of standard
0.572 g of disodium tetraborate R and 2.94 g of calcium pH.
ehlonde R in 800 rnL of water R . Adjust the pH (2.2.3) with Buffer Solution pH 5.5 Dissolve 54.4 g of sodiwl1
1 M hydrochlorie acid. Dilute to 1000.0 rnL with water R. acera te R in 50 rnL of water R, heating to 35 oC if necessary.
Borate Buffer Solution pH 10.4 Dissolve 24.64 g of boric After cooling, slowly add 10 rnL of anhydrous aeetie acid R.
acid R in 900 rnL of distilled water R . Adjust the pH (2.2.3) Shake and dilute to 100.0 rnL with water R.
using a 400 giL solution of sodium hydroxide R . Dilute to Buffer Solution pH 6.5 Dissolve 60.5 g of disodium
1000 rnL with distilled water R . hydrogen phosphate R and 46 g of potassiwn dihydrogen
Boric Buffer pH 9.0 Buffer solution pH 9.0 R1 phosphate R in water R . Add 100 rnL of 0.02 M sodium
edetate and 20 rng of mereurie chlOlide R and dilute to
Dissolve 6.20 g of bmic acid R in 500 rnL of water R and
1000.0 rnL with water R.
adjust the pH (2.2.3) with 1 M sodium hydroxide (about
41.5 rnL). Dilute to 1000.0 rnL with water R.
V-A152 Appendix 1 D 2014
Buffer Solution pH 6.6 To 250.0 rnL of 0.2 M potassium Citro-phosphate Buffer pH 5.0 Mix 48.5 rnL of
dihydrogen phosphate R add 89.0 rnL of 0.2 M sodium O.IM citric acid with sufficient 0.2M disodium hydrogen
hydroxide. Dilute to 1000.0 rnL with water R. orthophosphate to produce 100 roL.
Buffer Solution pH 7.0 To 1000 rnL of a solution Citro-phosphate Buffer pH 6.0 Phosphate buffer
containing 18 giL of disodium hydrogen phosphate R and solution pH 6.0.
23 giL of sodium chloride R add sufficient (about 280 rnL) of Mix 36.8 rnL of a 2.1 % w/v solution of cinic acid with
a solution containing 7.8 gIL of sodium dihydrogen 63.2 rnL of a 7.15% w/v solution of disodium hydrogen
phosphate R and 23 gIL of sodium chloride R to adjust the pH orthophosphate.
(2.2.3). Dissolve in the solution sufficient sodium azide R to Citro-phosphate Buffer pH 6.5 Mix 29.0 rnL of
give a 0.2 gIL solution. O.IM citric acidwith sufficient 0.2M disodium hydrogen
Buffer Solution pH 7.2 To 250.0 rnL of 0.2 M potassium orthophosphate to produce 100 rnL.
dihydrogen phosphate R add 175.0 rnL of 0.2 M sodium Citro-phosphate Buffer pH 6.8 Phosphate buffer
hydroxide. Dilute to 1000.0 rnL with water R. Adjust the pH solution pH 6.8
(2.2.3) if necessary.
Mix 77.3 rnL ofa 71.5 gIL solution of disodium hydrogen
Buffer Solution pH 7.4 Dissolve 0.6 g of potassium phosphate R with 22.7 rnL of a 21 gIL solution of citric acid R.
dihydrogen phosphate R, 6.4 g of disodium hydrogen
Citro-phosphate Buffer pH 7.0 Phosphate buffer
phosphate R and 5.85 g of sodium chloride R in water R, and
solution pH 7.0.
dilute to 1000.0 rnL with the sarne solvent. Adjust the pH
(2.2.3) ifnecessary. Mix 82.4 rnL of a 71.5 gIL solution of disodium hydrogen
phosphate R with 17.6 rnL of a 21 gIL solution of citric acid R.
Buffer Solution pH 8.0 To 50.0 rnL of 0.2 M potassium
dihydrogen phosphate R add 46.8 rnL of 0.2 M sodium Citro-phosphate Buffer pH 7.2 Phosphate buffer
hydroxide. Dilute to 200.0 rnL with water R. solution pH 7.2.
Buffer Solution pH 8.0 Rl Dissolve 20 g of dipotassium Mix 87.0 rnL of a 71.5 gIL solution of disodium hydrogen
hydrogen phosphate R in 900 rnL of water R. Adjust the pH phosphate R with 13.0 rnL of a 21 giL solution of citric acid R.
(2.2.3) with phosphoric acid R. Dilute to 1000 rnL with Citro-phosphate Buffer pH 7.6 Dissolve 67.1 g of
water R. disodium hydrogen orthophosphate and 1.33 g of citric acid in
Buffer Solution pH 9.0 Dissolve 6.18 g of boric acid R in sufficient water to produce 1000 rnL.
0.1 M potassium chlonde R and dilute to 1000.0 rnL with the Copper Sulfate Solution pH 2.0 Copper Sulphate
sarne solvent. Mix 1000.0 rnL of this solution and 420.0 rnL Solution pH 2.0.
of 0.1 M sodium hydroxide. Mix together 53 rnL of 0.2M hydrochloric acid, 250 rnL of
Buffer Solution pH 9.0 Rl Dissolve 6.20 g of boric acid R 0.2M potassium chloride and 40 rnL of a 0.393% w/v solution
in 500 rnL of water R and adjust the pH with 1 M sodium of copper(Il) sulfate and dilute with water to 1000 rnL.
hydroxide (about 41.5 rnL). Dilute to 1000.0 rnL with Copper Sulfate Solution pH 4.0, Buffered Copper
water R. Sulphate Solution pH 4.0, Buffered
Buffer Solution pH 10.9 Dissolve 6.75 g of ammonium Dissolve 0.25 g of copper sulfate R and 4.5 g of ammonium
chloride R in ammonia R and dilute to 100.0 rnL with the acetate R in dilute acetic acid R and dilute to 100.0 rnL with
sarne solvent. the sarne solvent.
Buffer Solution pH 11 Dissolve 6.21 g of boric acid R, Copper Sulfate Solution pH 5.2, Buffered Copper
4.00 g of sodium hydroxide R and 3.70 g of potassium Sulphate Solution pH 5.2, Buffered
chloride R in 500 roL of water R and dilute to 1000 roL with Dissolve 15.22 g of anhydrous disodium hydrogen
the sarne solvent. orthophosphate in sufficient water to produce 536 rnL and add
Buffered Salt Solution pH 7.2 Dissolve in water R 8.0 g a 2.1 % w/v solution of citric acid until the pH of the solution
of sodium chloride R, 0.2 g of potassium chloride R, 0.1 g of is between 5.15 and 5.25 (about 464 rnL). Mix 985 rnL of
anhydrous calcium chloride R, 0.1 g of magnesium chloride R, the resulting solution with 15 rnL of a 0.393% w/v solution
3.18 g of disodium hydrogen phosphate R and 0.2 g of of copper(Il) sulfate.
potassium dihydrogen phosphate R and dilute to 1000.0 rnL Deuterated Sodium Phosphate Buffer Solution pH 5.0,
with water R . 0.2M Dissolve 2.76 g of sodium dihydrogen phosphate
Carbonate Buffer pH 9.7 Dissolve 8.4 g of sodium monohydrate R in 90 rnL of deuterium oxide R, adjust the
hydrogen carbonate and 10.6 g of sodium carbonate in sufficient pH with a deuterated solution of phosphoric acid R or a
water to produce 50.0 rnL. deuterated 1 M solution of sodium hydroxide R, dilute to
ChIoride Buffer pH 2.0, O.IM Buffer solution pH 2.0 100 rnL with deuterium oxide R and rnix.
Dissolve 6.57 g of potassium chloride in water, add 119.O rnL Diethanolamine Buffer SoIution pH 10.0 Dissolve
of O.IM hydrochloric acid VS and dilute to 1000 rnL with 96.4 g of diethanolamine R in water R and dilute to 400 rnL
water. with the sarne solvent. Add 0.5 rnL of an 186 gIL solution of
Citrate Buffer Solution pH 5.0 Prepare a solution magnesium chloride R and adjust the pH (2.2.3) with 1 M
containing 20.1 gIL of citric acid R and 8.0 gIL of sodium hydrochloric acid. Dilute to 500.0 rnL with water R.
hydroxide R . Adjust the pH with dilute hydrochloric acid R. Diethylammonium Phosphate Buffer SoIution pH 6.0
Citrate Buffer Solution pH 3.0, 0.25M Dissolve 5.3 g of Dilute 68 rnL of phosphoric acid R to 500 rnL with water R.
citric acid R in 80 rnL of water R. Adjust the pH (2.2.3) with To 25 rnL of this solution add 450 rnL of water R and 6 rnL
1 M sodium hydroxide and dilute to 100.0 rnL with water R. of diethylamine R, adjust to pH 6 ± 0.05 (2.2.3), if
necessary, using diethylamine R or phosphoric acid R and
Citro-phosphate Buffer pH 4.5 To 30 volumes of
0.2M disodium hydrogen orthophosphate add sufficient dilute to 500.0 rnL with water R.
O.IM cinic acid to give a pH of 4.5 (about 36 volumes).
2014 Appendix 1 D V-A153
Glycine Buffer pH 2.9 Dissolve 6.0 g of glycine and dihydrogen orthophosphate in sufficient water to produce
4.68 g of sodium chloride in 10 litres of water. Adjust the pH 1000 mL and adjust the pH with glacial acetic acid.
with 1M hydrochlon:c acid (about 30 mL). Phosphate Buffer pH 4.75 Dilute 100 mL of
Glycine Buffer pH 11.3 Mix a solution containing 0.5M potassium dihydrogen orthophosphate to 800 mL with
0.75% w/v of glycine and 0.58% w/v of sodiUln chloride with water, adjust to pH 4.75 with O.IM sodium hydroxide and
an equal volume of O.IM sodium hydroxide and adjust the pH dilute to 1000 mL with water.
if necessary. Phosphate Buffer pH 4.9 Dissolve 40 g of sodium
Glycine Buffer Solution Mix 42 g of sodium hydrogen dihydrogen orthophosphate and 1.2 g of sodium hydroxide in
carbonate and 50 g of potassium hydrogen carbonate with sufficient water to produce 100 mL. If necessary, adjust the
180 mL of water and add a solution containing 37.5 g of pH with 1M sulfun'c acid or 1M sodium hydroxide as required.
glycine and 15 mL of 13.5M ammonia in 180 mL of water. Phosphate Buffer pH 5.4, Mixed Dissolve 1.76 g of
Dilute to 500 mL with water and stir until solution is disodium hydrogen orthophosphate and 13.61 g of potassium
complete. dihydrogen orthophosphate in sufficient water to produce
lnúdazole Buffer Solution pH 6.5 Dissolve 6.81 g of 1000 mL. Adjust the pH with 0.05M orthophosphoric acid, if
imidazole R, 1.23 g of magnesium sulfate R and 0.73 g of necessary.
calcium sulfate R in 752 mL of 0. 1 M hydrochloric acid. Adjust Phosphate Buffer pH 6.8, Mixed Dissolve 28.80 g of
the pH (2.2.3) ifnecessary and dilute to 1000.0 mL with disodium hydrogen orthophosphate and 11.45 g of potassium
water R . dihydrogen orthophosphate in sufficient water to produce
Inúdazole Buffer Solution pH 7.3 Dissolve 3.4 g of 1000 mL.
imidazole R and 5.8 g of sodium chloride R in water R, add Phosphate Buffer pH 6.8, 0.2M Mixed Phosphate buffer
18.6 mL of 1 M hydrochloric acid and dilute to 1000.0 mL solution pH 6.8 Rl.
with water R . Adjust the pH (2.2.3) if necessary.
Mix 51 mL of a 2.72% w/v solution of potassium d¡hydrogen
Maleate Buffer Solution pH 7.0 Dissolve 10.0 g of orthophosphate with 49 mL of a 7.16% w/v solution of
sodium chloride R, 6.06 g of disodium hydrogen orthophosphate and adjust the pH if
tris(hydroxymethyl)aminomethane R and 4.90 g of maleic necessary.
anhydride R in 900 mL of water R. Adjust the pH (2.2.3) 0
Store at 2° to 8 •
using a 170 gil solution of sodium hydroxide R. Dilute to
1000.0 mL with water R. Phosphate Buffer pH 7.0, Mixed Dissolve 0.50 g of
anhydrous disodium hydrogen orthophosphate and 0.301 g of
Storage: at 2 oC to 8 oC; use within 3 days.
potassium dihydrogen orthophosphate in sufficient water to
Octylamine Phosphate Buffer pH 3.0 Dilute 3.32 mL produce 1000 mL.
of octylamine to 1000 mL, adjust the pH to 3.0 using
Phosphate Buffer pH 7.0, 0.067M Mixed 0.067M
orthophosphoric acid and filter through a membrane filter with Phosphate buffer solution pH 7.0.
a nominal pore size not greater than 0.5 !lm.
Dissolve 0.908 g of potassium dihydrogen phosphate R in
Phosphate Buffers Solutions from pH 5.8 to pH 8.0 may
water R and dilute to 100.0 mL with the same solvent
be prepared by mixing 50 mL of 0.2M potassium dihydrogen
(solution A) . Dissolve 2.38 g of disodium hydrogen phosphate R
orthophosphate with the quantities of 0.2M sodium hydroxide VS
in water R and dilute to 100.0 mL with the same solvent
specified in the following table and diluting to 200 mL with
(solution B) . Mix 38.9 mL of solution A and 61.1 mL of
water. solution B. Adjust the pH (2.2.3) ifnecessary.
At 20° the solutions may be used as solutions of standard
Phosphate Buffer pH 7.0, O.IM Mixed O.IM Phosphate
pH. buffer solution pH 7.0
Dissolve 1.361 g of potassium dihydrogen phosphate R in
pH 5.8 6.0 6.2 6.4 6.6 6.8 water R and dilute to 100.0 mL with the same solvent.
mi ofO.2M sodium 3.72 5.70 8.60 12.60 17.80 23.65 Adjust the pH (2.2.3) using a 35 gIL solution of disodium
hydroxide VS hydrogen phosphate R.
pH 7.0 7.2 7.4 7.6 7.8 8.0 Phosphate Buffer pH 7.5, 0.2M 0.2M Phosphate buffer
mi ofO.2M sodíum 29.63 35.00 39.50 42.80 45.20 46.80 solution pH 7.5.
hydroxide VS Dissolve 27 .22 g of potassium dihydrogen phosphate R in
930 mL of water R, adjust to pH 7.5 (2.2.3) with a 300 gIL
solution of potassium hydroxide R and dilute to 1000.0 mL
Phosphate Buffer pH 3.0 Dissolve 34 g of potassium with water R .
dihydrogen orthophosphate in 250 mL of water and adjust the Phosphate Buffer pH 10, Mixed To 100 mL of
pH of the solution to 3.0 with orthophosphoric acid.
0.2M disodium hydrogen orthophosphate add 6.0 mL of
Phosphate Buffer pH 3.5 Phosphate buffer solution 0.25M trisodium orthophosphate.
pH 3.5.
Phosphate Buffer, 0.025M Standard Dissolve 3.40 g of
Dissolve 68 g of potassium dihydrogen orthophosphate in potassium dihydrogen orthophosphate and 3.55 g of anhydrous
1000 mL of water and adjust the pH of the solution to 3.5 disodium hydrogen orthophosphate, both previously dried at
with orthophosph011'C acid. 0
110' to 130 for 2 hours, in sufficient water to produce
Phosphate Buffer pH 4.0 Dissolve 6.8 g of potassium 1000 mL.
dihydrogen orthophosphate in 700 mL of water, adjust the pH, Phosphate Buffer Solution pH 2.0 Dissolve 8.95 g of
if necessary, with a 10% v/v solution of orthophosphol1'C acid disodium hydrogen phosphate R and 3.40 g of potassium
and add sufficient water to produce 1000 mL. dihydrogen phosphate R in water R and dilute to 1000.0 mL
Phosphate Buffer pH 4.0, Mixed Dissolve 5.04 g of with the same solvento If necessary adjust the pH (2.2.3)'
disodium hydrogen orthophosphate and 3.01 g of potassium with phosphOJic acid R .
Phosphate Buffer Solution pH 2.8 Dissolve 7.8 g of 1000.0 mL with water R. Adjust the pH (2.2.3) with strong
sodium dihydrogen phosphate R in 900 mL of water R, adjust sodium hydroxide solution R.
to pH 2.8 (2.2.3) with phosphoric acid R and dilute to Phosphate Buffer Solution pH 6.0 R2 To 250.0 mL of
1000 mL with the same solvent. 0.2 M potassium dihydrogen phosphate R add 28.5 mL of
Phosphate Buffer Solution pH 3.0 Mix 0.7 mL of 0.2 M sodium hydroxide and dilute to 1000.0 mL with
phosphoric acid R with 100 mL of water R. Dilute to 900 mL water R.
with the same solvent. Adjust to pH 3.0 (2.2.3) with strong Phosphate Buffer Solution pH 6.3, O.IM Dissolve 15 .6 g
sodium hydroxide solution R and dilute to 1000 mL with of sodium dihydrogen orthophosphate in 900 mL of water,
water R . adjust the pH to 6.3 with O.IMsodium hydroxide and add
Phosphate Buffer Solution pH 3.0, O.IM Dissolve 12.0 g sufficient water to produce 1000 mL.
of anhydrous sodium dihydrogen phosphate R in water R, adjust Phosphate Buffer Solution pH 6.4 Dissolve 2.5 g of
the pH (2.2.3) with dz1ute phosphoric acid Rl and dilute to disodium hydrogen phosphate R, 2.5 g of sodium dihydrogen
1000 mL with water R. phosphate R and 8.2 g of sodium chloride R in 950 mL of
Phosphate Buffer Solution pH 3.0 Rl Dissolve 3.40 g of water R. Adjust the pH (2.2.3) ofthe solution to 6.4 with
potassium dihydrogen phosphate R in 900 mL of water R. 1 M sodium hydroxide or 1 M hydrochloric acid, if necessary.
Adjust to pH 3.0 (2.2.3) with phosphoric acid R and dilute to Dilute to 1000.0 mL with water R.
1000.0 mL with water R. Phosphate Buffer Solution pH 6.5 Dissolve 2.75 g of
Phosphate Buffer Solution pH 3.2 To 900 mL of a sodium dihydrogen phosphate R and 4.5 g of sodium chloride R
4 gIL solution of sodium dihydrogen phosphate R, add 100 mL in 500 mL of water R. Adjust the pH (2.2.3) with phosphate
of a 2.5 gIL solution of phosphoric acid R. Adjust the pH buffer solution pH 8.5 R .
(2.2.3) if necessary. Phosphate Buffer Solution pH 6.5, O.IM Dissolve
Phosphate Buffer Solution pH 3.2 Rl Adjust a 35.8 gIL 13.80 g of sodium dihydrogen phosphate monohydrate R in
solution of disodium hydrogen phosphate R to pH 3.2 (2.2.3) 900 mL of distilled water R. Adjust the pH (2.2.3) using a
with dilute phosphoric acid R . Dilute 100.0 mL of the solution 400 gIL solution of sodium hydroxide R. Dilute to 1000 mL
to 2000.0 mL with water R . with distilled water R.
Phosphate Buffer Solution pH 3.5 Dissolve 68.0 g of Phosphate Buffer Solution pH 6.8 Mix 77.3 mL of a
potassium dihydrogen phosphate R in water R and dilute to 71. 5 gIL solution of disodium hydrogen phosphate R with
1000.0 mL with the same solvent. Adjust the pH (2.2.3) 22.7 mL of a 21 gIL solution of citric acid R.
with phosphoric acid R . Phosphate Buffer Solution pH 6.8 Rl To 51.0 mL of a
Phosphate Buffer Solution pH 4.5, 0.05M Dissolve 27.2 gIL solution of potassium dihydrogen phosphate R add
6.80 g of potassium dihydrogen phosphate R in 1000.0 mL of 49.0 mL of a 7l.6 gIL solution of disodium hydrogen
water R . The pH (2.2.3) of the solution is 4.5. phosphate R . Adjust the pH (2.2.3) if necessary.
Phosphate Buffer Solution pH 5.0 Dissolve 2.72 g of Storage: at 2 oC to 8 oc.
potassium dihydrogen phosphate R in 800 mL of water R. Phosphate Buffer Solution pH 7.0 Mix 82.4 mL of a
Adjust the pH (2.2.3) with 1 M potassium hydroxide and 71.5 giL solution of disodium hydrogen phosphate R with
dilute to 1000 mL with water R. 17.6 mL of a 21 gIL solution of citn'c acid R.
Phosphate Buffer Solution pH 5.4, 0.067M Mix Phosphate Buffer Solution pH 7.0 Rl Mix 250.0 mL of
appropriate volumes of a 23.99 gil solution of disodium 0.2 M potassium dihydrogen phosphate R and 148.2 mL of a
hydrogen phosphate R with a 9.12 gIL solution of sodium 8 gil solution of sodium hydroxide R, adjust the pH (2.2.3) if
dihydrogen phosphate monohydrate R to obtain pH 5.4 (2.2.3). necessary. Dilute to 1000.0 mL with water R.
Phosphate Buffer Solution pH 5.5 Dissolve 13.6 1 g of Phosphate Buffer Solution pH 7.0 R2 Mix 50.0 mL of a
potassium dihydrogen phosphate R in water R and dilute to 136 gIL solution of potassium dihydrogen phosphate R with
1000.0 mL with the same solvent (solution A). Dissolve 29.5 mL of 1 M sodium hydroxide and dilute to 100.0 mL
35.81 g of disodium hydrogen phosphate R in water R and with water R . Adjust the pH (2.2.3) to 7.0 ± 0.1,
dilute to 1000.0 mL with the same solvent (solution B). Appendix V L.
Mix 96.4 mL of solution A and 3.6 mL of solution B.
Phosphate Buffer Solution pH 7.0 R3 Dissolve 5 g of
Phosphate Buffer Solution pH 5.6 Dissolve 0.908 g of potassium dihydrogen phosphate R and 11 g of dipotassium
potassium dihydrogen phosphate R in water R and dilute to hydrogen phosphate R in 900 mL of water R. Adjust to pH 7.0
100.0 mL with the same solvent (solution A). Dissolve (2.2.3) with dz1ute phosphoric acid R or dilute sodium hydroxide
1.161 g of dipotassium hydrogen phosphate R in water R and solution R. Dilute to 1000 mL with water R and mix.
dilute to 100.0 mL with the same solvent (solution B).
Phosphate Buffer Solution pH 7.0 R4 Dissolve 28.4 g of
anhydrous disodium hydrogen phosphate R and 18.2 g of
If necessary, adjust to pH 5.6 (2.2.3) using solution A or
potassium dihydrogen phosphate R in water R and dilute to
solution B.
500 mL with the same solvent.
Phosphate Buffer Solution pH 5.8 Dissolve 1.19 g of
Phosphate Buffer Solution pH 7.0 R5 Dissolve 28.4 g of
disodium hydrogen phosphate dihydrate R and 8.25 g of
anhydrous disodium hydrogen phosphate R in 800 mL of
potassium dihydrogen phosphate R in water R and dilute to
water R . Adjust the pH (2.2.3) using a 30 per cent mlm
1000.0 mL with the same solvent.
solution of phosphoric acid R and dilute to 1000 mL with
Phosphate Buffer Solution pH 6.0 Mix 63.2 mL of a water R.
71.5 giL solution of disodium hydrogen phosphate R and
Phosphate Buffer Solution pH 7.0, 0.025M Mix
36.8 mL of a 21 gIL solution of citric acid R.
1 volume of 0.063 M phosphate buffer solution pH 7. O R with
Phosphate Buffer Solution pH 6.0 Rl Dissolve 6.8 g of 1.5 volumes of water R.
sodium dihydrogen phosphate R in water R and dilute to
2014 Appendix 1 D V-A155
Phosphate Buffer Solution pH 7.0, 0.03M Dissolve Phosphate-Citrate Buffer Solution pH 5.5 Mix
5.2 g of dipotassium hydrogen phosphate R in 900 mL of water 56.85 rnL of a 28.4 giL solution of anhydrous disodium
for chromatography R. Adjust the solution to pH 7.0 ± 0.1 hydrogen phosphate R and 43.15 rnL of a 21 giL solution of
using phosphoric acid R and dilute to 1000 mL with water for citric acid R.
chromatography R. Phthalate Buffer pH 3.6 Buffer solution pH 3.6
Phosphate Buffer Solution pH 7.0, 0.05M Mix 34 mL To 250 rnL of 0.2M potassium hydrogen phthalate add
of water R and 100 mL of 0.067 M phosphate buffer solution 11.94 mL of 0.2M hydrochloric acid VS and dilute to
pH 7.0 R. 1000 rnL with water.
Phosphate Buffer Solution pH 7.0, 0.063M Dissolve At 20°, the solution rnay be used as a solution of standard
5.18 g of anhydrous disodium hydrogen phosphate R and 3.65 g pH.
of sodium dihydrogen phosphate monohydrate R in 950 mL of Phthalate Buffer Solution pH 4.4 Dissolve 2.042 g of
water R and adjust the pH (2.2.3) with phosphoric acid R; potassium hydrogen phthalate R in 50 rnL of water R, add
dilute to 1000.0 mL with water R. 7.5 rnL of 0.2 M sodium hydroxide and dilute to 200 .0 mL
Phosphate Buffer Solution pH 7.0, 0.067M Dissolve with water R .
0.908 g of potassium dihydrogen phosphate R in water R and At 20°, the solution may be used as a solution of standard
dilute to 100.0 mL with the same solvent (solution A). pH.
Dissolve 2.38 g of disodium hydrogen phosphate R in water R
Phthalate Buffer Solution pH 6.4, 0.5M Dissolve 100 g
and dilute to 100.0 mL with the same solvent (solution B).
of potassium hydrogen phthalate R in water R and dilute to
1000.0 rnL with the sarne solvento Adjust the pH (2.2.3) if
Adjust the pH if necessary.
necessary, using strong sodium hydroxide solution R.
Phosphate Buffer Solution pH 7.0, O.IM Dissolve
Saline pH 6.4, Phosphate-buffered Dissolve 1.79 g of
1.361 g of potassiwn dihydrogen phosphate R in water R and
disodium hydrogen orthophosphate, 1.36 g of potassiwn
dilute to 100.0 mL with the same solvento Adjust the pH
dihydrogen orthophosphate and 7.02 g of sodium chloride in
using a 35 gIL solution of disodium hydrogen phosphate R.
sufficient water to produce 1000 rnL.
Phosphate Buffer Solution pH 7.2 Mix 87.0 mL ofa
Saline pH 6.8, Phosphate-buffered Dissolve 1.0 g of
71.5 giL solution of disodium hydrogen phosphate R with
potassium dihydrogen phosphate R, 2.0 g of dipotassium
13.0 mL of a 21 gIL solution of citric acid R.
hydrogen phosphate R and 8.5 g of sodium chloride R in
Phosphate Buffer Solution pH 7.4 Add 250.0 mL of 900 rnL of water R, adjust the pH (2.2.3) if necessary and
0.2 M potassium dihydrogen phosphate R to 393.4 mL of dilute to 1000.0 rnL with the same solvento
0.1 M sodium hydroxide.
Saline pH 7.2, Phosphate-alburnin Buffered Dissolve
Phosphate Buffer Solution pH 7.5, 0.2M Dissolve 10.75 g of disodium hydrogen phosphate R, 7.6 g of sodium
27.22 g of potassium dihydrogen phosphate R in 930 rnL of chloride R and 10 g of bovine albumin R in water R and dilute
water R, adjust to pH 7.5 with a 300 gIL solution of to 1000.0 rnL with the same solvent. Irnmediately before use
potassium hydroxide R and dilute to 1000.0 mL with water R. adjust the pH (2.2.3) using dilute sodium hydroxide solution R
Phosphate Buffer Solution pH 7.5, 0.33M Dissolve or dilute phosphoric acid R.
119.3 1 g of disodium hydrogen phosphate R in water R and Saline pH 7.2 Rl, Phosphate-alburnin Buffered
dilute to 1000.0 rnL with the same solvent (solution A). Dissolve 10.75 g of disodium hydrogen phosphate R, 7.6 g of
Dissolve 45.36 g of potassium dihydrogen phosphate R in sodium chloride R and 1 g of bovine albumin R in water R and
water R and dilute to 1000.0 mL with the sarne solvent dilute to 1000.0 mL with the same solvento Immediately
(solution B). Mix 85 mL of solution A and 15 mL of before use adjust the pH (2.2.3) using dilute sodium hydroxide
solution B. Adjust the pH (2.2.3) if necessary. solution R or dilute phosphoric acid R.
Phosphate Buffer Solution pH 8.0, 0.02M To 50.0 rnL Saline pH 7.4, Phosphate-buffered Dissolve 2.38 g of
of 0.2 M potassium dihydrogen phosphate R add 46.8 rnL of disodium hydrogen phosphate R, 0.19 g of porassium dihydrogen
0.2 M sodium hydroxide. Dilute to 500.0 mL with water R. phosphate R and 8.0 g of sodium chloride R in water. Dilute to
Phosphate Buffer Solution pH 8.0, O.IM Dissolve 1000.0 rnL with the same solvento Adjust the pH (2.2.3) if
0.523 g of potassium dihydrogen phosphate R and 16.73 g of necessary.
dipo¡assium hydrogen phosphate R in water R and dilute to Sodium Acetate Buffer pH 7.0 Dissolve 1.64 g of
1000.0 rnL with the same solvento anhydrous sodium acetate in 1 L of water, adjust to pH 7.0
Phosphate buffer solution pH 8.0, 0.05 M Dissolve using dilute acetic acid.
0.31 g of sodium dihydrogen phosphate R in 70 rnL of water R Sodium Acetate Buffer Solution pH 4.0, 0.1 M Dissolve
and adjust to pH 8.0 with 1 M sodium hydroxide, then dilute 822 rng of sodium acetate R in 100 mL of water R
to 100 rnL with water R . (solution A). Dilute 1.44 rnL of glacial acetic acid R in
Phosphate Buffer Solution pH 8.0, 1M Dissolve 136.1 g 250 rnL of water R (solution B). Titrate 100 mL of
of potassium dihydrogen phosphate R in water R, adjust the pH solution B using about 20 mL of solution A.
(2.2.3) with 1 M sodium hydroxide. Dilute to 1000.0 mL with Sodium Acetate Buffer Solution pH 4.5 Dissolve 63 g
water R. of anhydrous sodium acerate R in water R, add 90 rnL acetic
Phosphate Buffer Solution pH 8.5 Dissolve 3.5 g of acid R and adjust to pH 4.5, and dilute to 1000 mL with
dipotassium hydrogen phosphate R and 4.5 g of sodium water R .
chloride R in 500 mL of water R. Adjust the pH (2.2.3) with Sodium Acetate Solution pH 6.0, Buffered Dissolve
a mixture of equal volurnes of dilute phosphoric acid R and 4.1 g of anhydrous sodium acetate in 1000 rnL of water and
water R. adjust the pH to 6.0 with glacial acetic acid.
Phosphate Buffer Solution pH 9.0 See buffer (phosphate) Sodium Citrate Buffer Solution pH 7.8 (0.034M Sodium
solution pH 9. O. Citrate, 0.101M Sodium Ch1oride) Dissolve 10.0 g of
sodium citrate R and 5.90 g of sodium chloride R in 900 rnL of
water R . Adjust the pH (2.2.3) by addition of hydrochloric Tris-chloride Buffer pH 7.5 R1 0.05M Tris-
acid R and dilute to 1000 mL with water R. hydrochloride buffer solution pH 7.5
Sodium Phosphate Buffer Solution pH 8.0, 0.02M Dissolve 6.057 g of tris(hydroxymethyl)aminomethane R in
Dissolve 0.31 g of sodium dihydrogen phosphate R in 70 mL water R and adjust the pH (2.2.3) with hydrochloric acid R.
of water R and adjust to pH 8.0 with 1 M sodium hydroxide, Dilute to 1000.0 mL with water R .
then dilute to 100 mL with water R. Tris-chloride Buffer pH 8.1
Succinate Buffer Solution pH 4.6 Disssolve 11.8 g of Tris(hydroxymethyl)aminomethane buffer solution pH 8.1.
succinic acid R in a mixture of 600 mL of water R and 82 mL Dissolve 0.294 g of calcium chloride and 0.969 g of
of 1 M sodium hydroxide and dilute to 1000.0 mL with tris(hydroxymethyl)methylamine in water, adjust the pH to 8.1
water R. with 1M hydrochloric acid and add sufficient water to produce
Sulfate Buffer Solution pH 2.00 Sulphate Buffer 100 mL.
Solution pH 2.0 Tris-chloride Buffer pH 8.6 Dissolve 2.0 g of
Dissolve 132.1 g of ammonium sulfate R in water R and dilute tris(hydroxymethyl)methylamine and 2.4 g of sodium chloride in
to 500.0 mL with the same solvent (Solution A) . Carefully about 100 mL of water, adjust the pH to 8.6 with 1M sodium
and with constant cooling stir 14 mL of sulfuric acid R into hydroxide or 1M hydrochlorie acid and dilute with water to
about 400 mL of water R; allow to cool and dilute to 200 mL.
500.0 mL with water R (Solution B). Mix equal volumes of Tris-EDTA Buffer pH 8.4
solutions A and B. Adjust the pH (2.2.3) if necessary. Tris(hydroxymethyl)aminomethane EDTA buffer solution
Tetrabutylammonium Buffer Solution pH 7.0 Dissolve pH 8.4
6.16 g of ammonium aceta te R in a mixture of 15 mL of Dissolve 5.12 g of sodium chloride R, 3.03 g of
tetrabut:ylammonium hydroxide solution (400 giL) R and tris(hydroxymethyl)aminomethane R and 1.40 g of sodium
185 mL of water R. Adjust the pH (2.2.3) with nitric acid R . edetate R in 250 mL of distilled water R. Adjust the pH
Thiobarbiturie Acid-citrate Buffer Dissolve 5.0 g of (2.2.3) to 8.4 using hydroehloric acid R. Dilute to 500.0 mL
thiobarbituric acid in 5 mL of 4M sodium hydroxide and dilute with distilled water R .
to 500 mL with water. Dissolve separately 37 g of sodium Tris-EDTA BSA Buffer Solution pH 8.4 Dissolve 6.1 g
citrate in 32 roL of hydrochloric acid and dilute to 250 mL of tris(hydroxymethyl)aminomethane R, 2.8 g of sodium
with water. Mix the two solutions and adjust the pH of the edetate R, 10.2 g of sodium chloride R and 10 g of bovine
resulting solution to 2.0. albumin R in water R, adjust to pH 8.4 (2.2.3) using 1 M
Total Ionie Strength Adjustment Buffer Dissolve 58.5 g hydrochloric acid and dilute to 1000.0 mL with water R.
of sodium chloride R, 57.0 mL of glacial acetic acid R , 61.5 g Tris-glycine Buffer Solution pH 8.3 Dissolve 6.0 g of
of sodium acetate R and 5.0 g of cyclohexylene-dinitrilotetra- tris(hydroxymethyl)aminomethane R and 28.8 g of glycine R in
acetic acid R in water R and dilute to 500.0 mL with the water R and dilute to 1000.0 mL with the same solvento
same solvento Adjust to pH 5.0 to 5.5 with a 335 gIL Dilute 1 volume to 10 volumes with water R immediately
solution of sodium hydroxide R and dilute to 1000.0 mL with before use.
distilled water R.
Tris-hydroch1oride Buffer Solution pH 6.8, 1M
Total Ionie Strength Adjustment Buffer R1 Dissolve 60.6 g of tris(hydroxymethyl)aminomethane R in
Dissolve 210 g of citric acid R in 400 mL of distilled water R. 400 mL of water R. Adjust the pH (2.2.3) with hydrochloric
Adjust to pH 7.0 (2.2.3) with concentrated ammonia R . Dilute acid R and dilute to 500.0 mL with water R.
to 1000.0 mL with distilled water R (solution A). Dissolve Tris-hydroch1oride Buffer Solution pH 7.5, 0.05M
132 g of ammonium phosphate R in distilled water R and dilute Dissolve 6.057 g of tris(hydroxymethyl)aminomethane R in
to 1000.0 mL with the same solvent (solution B). To a water R and adjust the pH with hydroehloric acid R. Dilute to
suspension of 292 g of (ethylenedinitrilo)tetra-acetic acid R in 1000.0 mL with water R .
about 500 mL of distilled water R, add about 200 mL of
Tris-hydroch1oride Buffer Solution pH 8.0 Dissolve
concentrated ammonia R to dissolve. Adjust the pH to 6 to 7
1.21 g of tris(hydroxymethyl)aminomethane R and 29.4 mg of
(2.2.3) with concentrated ammonia R. Dilute to 1000.0 mL
ealcium ehloride R in water R. Adjust the pH (2.2.3) with
with distilled water R (solution C). Mix equal volumes of
1 M hydroehloric ¡¡cid and dilute to 100.0 mL with water R.
solution A, B, and C and adjust to pH 7.5 with coneentrated
ammonia R .
Tris-hydroch1oride Buffer Solution pH 8.0, 1M
Dissolve 121.1 g of tris (hydroxymethyl) aminomethane R and
Tris-acetate Buffer Solution pH 8.5 Dissolve 0.294 g of
1.47 g of ealcium ehlonde R in 900 mL of water R. Adjust the
calcium chloride R and 12.11 g of
pH (2.2.3) with hydroehlorie acid R and dilute to 1000.0 mL
tris(hydroxymethyl)aminomethane R in water R. Adjust the pH
with water R.
(2.2.3) with acetic acid R. Dilute to 1000.0 mL with water R.
Tris-hydroch1oride Buffer Solution pH 8.3 Dissolve
Tris-ch1oride Buffer pH 7.4
9.0 g of tris(hydroxymethyl)aminomethane R in 2.9 litres of
Tris(hydroxyrnethyl)aminomethane sodium chloride buffer
water R. Adjust the pH (2. 2.3) with 1 M hydrochloric acid.
solution pH 7.4
Adjust the volume to 3 litres with water R .
Dissolve 6.08 g of tris(hydroxymethyl)aminomethane R, 8.77 g
Tris-hydroch1oride Buffer Solution pH 8.8, 1.5M
of sodium chloride R in 500 mL of distilled water R.
Dissolve 90.8 g of tris(hydroxymethyl)aminomethane R in
Add 10.0 g of bovine albumin R. Adjust the pH (2.2.3) using
400 mL of water R . Adjust the pH (2.2.3) with hydrochloric
hydrochloric acid R. Dilute to 1000.0 mL with distilled water R.
acid R and dilute to 500.0 mL with water R .
Tris-chloride Buffer pH 7.5
Tris-hydroch1oride Buffer Solution pH 9.0, 0.05M
Tris(hydroxymethyl)aminomethane buffer solution pH 7.5.
Dissolve 0.605 g of tris(hydroxymethyl)aminomethane R in
Dissolve 7.27 g of tris(hydroxymethyl)aminomethane R and water R. Adjust the pH (2.2.3) with 1 M hydrochloric acid
5.27 g of sodium chloride R in water R, and adjust the pH and dilute to 100.0 mL with water R.
(2.2.3) if necessary. Dilute to 1000.0 mL with water R.
2014 Appendix 1 F V-A157
Tris(hydroxymethyl)aminomethane Buffer Solution from the BPCRS Sales Office, MHRA, 5th Floor, 151
pH 7.4 Dissolve 30.3 g of Buckingham Palace Road, London SWIW 9SZ, United
tris(hydroxymethyl)aminomethane R in approximately 200 mL Kingdom (telephone +44 (0)20 3080 6561,
of water R. Add 183 mL of 1 M hydrochloric acid. Dilute to e-mail: bpcom@mhra.gsi.gov.uk.
500.0 mL with water R. website www.pharmacopoeia.com).
Note: The pH is 7.7-7.8 at room temperature and 7.4 at 37 oC. In addition to BPCRS, monographs of the British
This solution is stable for several months at 4 oC. Pharmacopoeia may also refer ro reference substances
Tris(hydroxymethyI)aminomethane Buffer Solution available from other suppliers. These are denoted by the
pH 7.5 Dissolve 7.27 g of letters CRS.
tris(hydroxymethyl)aminomethane R and 5.27 g of sodium Where the letters CRS or EPCRS appear, the chemicaJ
chloride R in water R, and adjust the pH if necessary. Dilute reference substance issued by the European Pharmacopoeia
to 1000.0 mL with water R. Commission is to be used; where the letters BRP or EPBRP
Tris(hydroxymethyl)aminomethane Buffer Solution appear, the Biological Reference Preparation issued by the
pH 8.1 Dissolve 0.294 g of calcium chloride R in 40 mL of European Pharmacopoeia Commission is ro be used; where
tris(hydroxymethyl)aminomethane solution R and adjust the pH the letters HRS or EPHRS appear, the herbal reference
with 1 M hydrochloric acid. Dilute to 100.0 mL with water R. substance issued by the European Pharmacopoeia
Tris(hydroxymethyl)aminomethane-EDTA-Buffer Commission is ro be used. The substances, as well as
European Pharmacopoeia infrared reference spectra, are
Solution pH 8.4 Dissolve 5.12 g of sodium chloride R,
3.03 g of tris(hydroxymethyl)aminomethane R and 1.40 g of obtainable from the Council of Europe, European
sodium edetate R in 250 mL of distilled water R. Adjust the
Directorate for the Quality of Medicines & HealthCare, CRS
Sales Team, 7 allée Kastner, CS 30026, F-67081, Strasbourg
pH to 8.4 using hydrochloric acid R. Dilute to 500.0 mL with
Cedex, France (facsímile +33 (0)3 88 41 27 71,
distilled water R.
e-mail: crs@pheur.org, website www.edqm.eu).
Tris(hydroxymethyI)aminomethane Sodium ChIoride
Other sources of specific reference substances are shown
Buffer solution pH 7.4 See tris-chloride Buffer pH 7.4.
below.
Tris(hydroxymethyl)aminomethane Sodium ChIoride
Buffer Solution pH 7.4 R1 Dissolve 0.1 g of bovine Astragaloside 1 CRS, Astragaloside n CRS,
Astragaloside IV CRS, azadirachtin A CRS, bacopaside
albumin R in a mixture containing 2 mL of
1 CRS, bacopaside 11 CRS, bacoside A CRS,
tris(hydroxymethyl)aminomethane buffer solution pH 7.4 R and
PaeonoI CRS, Rosmarinic acid CRS, salannin CRS,
50 mL of a 5.84 mglmL solution of sodium chloride R. Dilute
Salvianolic Acid B CRS, Tanshinone nA CRS,
to 100.0 mL with water R.
Withaferin A CRS, Withanolide ACRS, Withanolide
Tris-sodium Acetate Buffer Solution pH 7.4 Dissolve B CRS, Z-Ligustilide CRS may be obtained from
6.3 g of tris(hydroxymethyl)aminomethane R and 4.9 g of Chromadex Inc. through LGC Standards, Queen's Road,
anhydrous sodium acetate R in 900 mL of water R. Adjust to Teddingron, TWll OLY, United Kingdom
pH 7.4 (2.2.3) with sulfuric acid R and dilute ro 1000 mL (telephone +44 (0)20 8943 8480,
with water R. facsimile +44 (0)20 8943 7554,
Tris-sodium Acetate Buffer Solution pH .8.0 Dissolve e-mail: uksales@lgcstandards.com).
6.3 g of tris(hydroxymethyl)aminomethane R and 4.9 g of Opacity, Standard Preparation of The Standard
anhydrous sodium acetate R in 900 mL of water R. Adjust to Preparation is the 5th Intemational Reference Preparation,
pH 8.0 (2.2.3) with sulfuric acid R and dilute to 1000 mL established in 1975, and consists of a rod of plastic
with water R. simulating the optical properties of a bacterial suspension (10
Tris-sodium Acetate-sodium ChIoride Buffer SoIution Units of opacity). It may be obtained from the National
pH 7.4 Dissolve 30.0 g of Institute for Biological Standards and Control (NIBSC),
tris(hydroxymethyl)aminomethane R, 14.5 g of anhydrous Blanche Lane, South Mímms, Potters Bar, Hertfordshire,
sodium acetate R and 14.6 g of sodium chloride R in 900 mL EN6 3QG, United Kingdom
of water R. Add 0.50 g of bovine albumin R. Adjust to (telephone +44 (O) 1707 641000,
pH 7.4 (2.2.3) with sulfuric acid R and dilute ro 1000 mL e-mail: enquiries@nibsc.org).
with water R. Piperonyl Butoxide CRS may be obtained from LGC
Tris-sodium Acetate-Sodium ChIoride Buffer Solution Standards, Queen's Road, Teddington, TWll OLY, United
pH 8.0 Dissolve 30.0 g of Kingdom (telephone +44 (0)20 8943 8480,
tris(hydroxymethyl)aminomethane R, 14.5 g of anhydrous facsimile +44 (0)20 8943 7554,
sodium acetate R and 14.6 g of sodium chloride R in 900 mL e-mail: uksales@lgcstandards.com).
of water R. Add 0.50 g of bovine albumin R. Adjust ro
pH 8.0 (2.2.3) with sulfuric acid R and dilute ro 1000 mL
with water R.
F. Polymorphism
(Ph. Eur. method 5.9)
E. Reference Materials Polymorphism (or crystal polymorphism) is a phenomenon
Where the letters BPCRS appear after the name of a related ro the solid sta te; it is the ability of a compound in
substance in a test or assay, the British Pharmacopoeia the solid state ro exist in different crystalline forms having the
Chemical Reference Substance is to be used. same chemical composition. Substances that exist in a non-
A comprehensive and up-ro-date list of British crystalline solid state are said to be amorphous.
Pharmacopoeia Chemical Reference Substances, together When this phenomenon is observed for a chemical eJement
with terms of trade and supply, is available on our website at (for example, sulfur), the term allotropy is used instead of
www.pharmacopoeia.com. The substances are obtainable polymorphism.
V-A158 Appendix 1 F 2014
The tenn pseudopolymorphism is used to describe solvates the energetic relationship (enantiotropism, monotropism) and
(inc1uding hydrates), where a solvent is present in the crystal the thermodynamic stability of the individual modifications of
matrix in stoichiometric proportions; the term may also be a polymorphic compound.
extended to include compounds where the solvent is trapped For solvates, differential scanning calorimetry and
in the matrix in variable proportions. However the tenn thennogravimetry are preferable, combined with
pseudopolymorphism is ambiguous because of its use in measurements of solubility, intrinsic dissolution rate and
different circumstances. It is therefore preferable to use only X-ray diffraction.
the tenns "solvates" and "hydrates". For hydrates, water sorptionldesorption isothenns are
Where a monograph indicates that a substance shows detennined to demonstrate the zones of relative stability.
polymorphism, this may be true crystal polymorphism, In general, hydrates are less soluble in water than anhydrous
occurence of solvates, allotropy or occurrence of the forms, and likewise solvates are less soluble in their solvent
amorphous formo than unsolvated fonns.
The identity of chemical composition implies that all
crystalline and amorphous forms of a given species have the
same chemical behaviour in solution or as a melt; in contrast,
their physico-chemical and physical characteristics (solubility,
hardness, compressibility, density, melting point, etc.), and
therefore their reactivity and bioavailability may be different
at the solid state .
When a compound shows polymorphism, the form for which
the free enthalpy is lowest at a given temperature and
pressure is the most thennodynamically stable. The other
forms are said to be in a metastable state. At nonnal
temperature and pressure, a metastable form may remain
unchanged or may change to a thermodynamically more
stable formo
If there are several crystalline forms, one form is
thennodynamically more stable at a given temperature and
pressure. A given crystalline form may constitute a phase that
can reach equilibrium with other solid phases and with the
liquid and gas phases.
If each crystalline fonn is the more stable within a given
temperature range, the change from one fonn to another is
reversible and is said to be enantiotropic. The change from
one phase to another is a univariate equilibrium, so that at a
given pressure this state is characterised by a transition
temperature. However, if only one of the forms is stable over
the entire temperature range, the change is irreversible or
monotropic.
Different crystalline forms or solvates may be produced by
varying the crystallisation conditions (temperature, pressure,
solvent, concentration, rate of crystallisation, seeding of the
crystallisation medium, presence and concentration of
impurities, etc .).
The following techniques may be used to study
polymorphism:
- X-ray diffraction of powders,
- X-ray diffraction of single crystals,
- thennal analysis (2.2.34) (differential scanning
calorimetry, thennogravimetry, thennomicroscopy),
- microcalorimetry,
- moisture absorption analysis,
- optical and electronic microscopy,
- solid-state nuclear magnetic re so nance,
- infrared absorption spectrophotometry (2.2.24),
- Raman spectrometry (2.2.48),
- measurement of solubility and intrinsic dissolution rate,
- density measurement.
These techniques are often complementary and it is
indispensable to use several of them.
Pressure/temperature and energy/temperature diagrams based
on analytical data are valuable tools for fully understanding
2014 Appendix JI A V-A159
Triturate 1-2 mg of the substance to be examined wíth

Appendix II 300-400 mg, unless otherwise specified, of finely
powdered and dried potassium bromide R or potassium
chloride R . These quantities are usually sufficient to give
A. Infrared Spectrophotometry a disc of 10-15 mm diameter and a spectrum of suitable
(Ph. Eur. method 2.2.24) intensity. If the substance is a hydrachloride, it is
Infrared spectrophotometers are used for recording spectra in recommended to use potassium chloride R. Carefully
the region of 4000-650 cm- 1 (2.5-15.4 Ilm) or in sorne cases grind the mixture, spread it uniformly in a suitable die,
down to 200 cm- 1 (50 Ilm) . and submit ir to a pressure of about 800 MPa (8
t·cm- 2). For substances that are unstable under normal
Apparatus atmospheric conditions or are hygroscopic, the disc is
Spectraphotometers for recording spectra consist of a suitable pressed in vacuo. Several factors may cause the
light source, monochromator or interferometer and detector. formation of faulty discs, such as insufficient or excessive
grinding, humidity or other impurities in the dispersion
Fourier transform spectrophotometers use polychromatic
medium or an insufficient reduction of partic1e size.
radiatíon and calculate the spectrum in the frequency domain
A disc is rejected if visual examination shows lack of
fram the original data by Fourier transformation.
uniform transparency or when transmittance at about
Spectrophotometers fitted with an optical system capable of
2000 cm- 1 (5 Ilm) in the absence of a specific
producing monochromatic radiation in the measurement
absorption band is less than 60 per cent without
region may also be used. Normally the spectrum is given as a
compensation, unless otherwise prescribed.
function of transmittance, the quotient of the intensity of the
transmitted radiation and the incident radiation. It may also Gases Examine gases in a cell transparent to infrared
be given in absorbance. radiation and having an optical path length of about
100 mm. Evacuate the cell and fill to the desired pressure
The absorbance (A) is defined as the logarithm to base 10 of
through a stopcock or needle valve using a suitable gas
the reciprocal of the transmittance (7):
transfer line between the cell and the container of the gas to
be examined.
A = loglo (~ ) = 10glO ( 7) If necessary adjust the pressure in the cell to atmospheric
pressure using a gas transparent to infrared radiation (for
example nitrogen R and argon R). To avoid absorption
1
T interferences due to water, carbon dioxide or other
lo' atmospheric gases, place in the reference beam, if possible,
lo intensity of incident radiation, an identical cell that is either evacuated or filled with the gas
1 intensity of transmitted radiation. transparent to infrared radiation.
For recording by diffuse reflectance
Preparation of the sample
Solids Triturate 11 mixture of the substance to be examined
For recording by transmission or absorption with finely powdered and dried potassium bromide R or
Prepare the substance by one of the following methods. potassium chloride R . Use a mixture containing approximately
Liquids Examine a liquid either in the form of a film 5 per cent of the substance, unless otherwise specified. Grind
between 2 plates transparent to infrared radiation, or in a the mixture, place it in a sample cup and examine the
cell of suitable path length, also transparent to infrared reflectance spectrum.
radiation. The spectrum of the sample in absorbance mode may be
Liquids or solids in solution Prepare a solution in a obtained after mathematical treatment of the spectra by the
suitable solvent. Choose a concentration and a path length of Kubelka-Munk function.
the cell which give a satisfactory spectrum. Generally, good For recording by attenuated total reflection
results are obtaíned with concentrations of 10-100 giL for a Attenuated total reflection (including multiple reflection)
path length of 0.5-0.1 mm. Absorption due to the solvent is involves light being reflected intemally by a transmitting
compensated by placing in the reference beam a similar cell medium, typically for a number of reflections . However,
containing the solvent used. If an FT-IR instrument is used, several accessories exist where only one reflection occurs.
the absorption is compensated by recording the spectra for
Prepare the substance as follaws . Place the substance to be
the solvent and the sample successively. The solvent
examined in clase contact with an intemal reflection element
absorbance, corrected by a compensation factor,is
(IRE) such as diamond, germanium, zinc selenide, thallium
subtracted using calculation software.
bromide-thallium iodide (KRS-5) ar another suitable material
Solids Examine solids dispersed in a suitable liquid (mull) of high refractive indexo Ensure clase and uniform contact
or in a salid (halide disc), as appropriate. If prescribed in the between the substance and the whole crystal surface of the
monograph, make a film of a mol ten mass between 2 plates intemal reflection element, either by applying pressure or by
transparent to infrared radiation. dissolving the substance in an appropriate solvent, then
A. Mull covering the IRE with the obtained solution and evaporating
Triturate a small quantity of the substance to be to dryness. Examine the attenuated total reflectance (ATR)
examined with the minimum quantity of liquid paraffin R spectrum.
or other suitable liquid; 5-10 mg of the substance to be
examined is usually sufficient to make an adequate mull Identification using reference substances
using one drap of liquid paraffin R. Compress the mull Prepare the substance to be examined and the reference
between 2 plates transparent to infrared radiation. substance by the same procedure and record the spectra
B. Disc between 4000-650 cm- 1 (2.5-15.4 Ilm) under the same
operational conditions. The transmission minima (absorption
V-A160 Appendix II A 2014
maxima) in tbe spectrum obtained witb the substance to be

examined correspond in position and relative size to tbose in
~ ~ft ~
the spectrum obtained witb the reference substance (CRS).
When tbe spectra recorded in tbe solid state show differences
80 80
in tbe positions of tbe transmission minima (absorption
maxima), treat tbe substance to be examined and the
reference substance in tbe same manner so tbat tbey
/
crystaJlise or are produced in tbe same form, or proceed as 60 60
prescribed in tbe monograph, tben record the spectra. e
Identification using reference spectra !
Control of resolution performance For instruments
having a monochromator, record the spectrum of a
40
A
40
1 D
polystyrene film approximately 35 [lm in thickness.
The difference x (see Figure 2.2.24.-1) between tbe
percentage transmittance at tbe transmission maximum A at 20 20
2870 cm- 1 (3.48 [lm) and tbat at tbe transrnission minimum
B at 2849.5 cm- 1 (3.51 [lm) must be greater tban 18. B
The difference y between the percentage transmittance at tbe
o o
transmission maximum C at 1589 cm- 1 (6.29 ~lm) and tbat 3200 3000 2800 2600 1800 1600 1400
at tbe transmission minimum D at 1583 cm- 1 (6.32 [lm) Wave number cm - 1
must be greater tban 10.
Table 2.2.24.-1. - Transmission minima and acceptable

Figure 2.2.24.-1. - Typical specfrum of polysfyrene used fo
toleran ces of a po[ystyrene film
verify the resolution performance
Transmission Acceptable tolerance (cm- l
)
mínima (cm- l ) Method Prepare the substance to be examined according
to tbe instructions accompanying tbe reference
Monochromator Fourier-transform
ínslrumenls ínstrumenls spectrum/reference substance. Using tbe operating conditions
tbat were used to obtain the reference spectrum, which wiJl
3060.0 ± 1.5 ± 1.0 usuaJly be tbe same as tbose for verifying the resolution
2849.5 ± 2.0 ± 1.0
performance, record the spectrum of tbe substance to be
examined.
1942.9 ± 1.5 ± 1.0 The positions and tbe relative sizes of tbe bands in tbe
1601.2 ± 1.0 ± 1.0
spectrum of tbe substance to be exarnined and tbe reference
spectrum are concordant in tbe 2 spectra.
1583.0 ± 1.0 ± 1.0
Compensation for water vapour and atmospheric
1154.5 ± 1.0 ± 1.0 carbon dióxide For Fourier-transform instruments,
spectral interference from water vapour and carbon dioxide
1028.3 ± 1.0 ± 1.0 is compensated using suitable algorithms according to tbe
manufacturer's instructions. Alternatively, spectra can be
acquired using suitable purged instruments or ensuring tbat
For Fourier-transform instruments, use suitable instrument
sample and background single beam spectra are acquired
resolution witb the appropriate apodisation prescribed by tbe
under exact1y tbe same conditions.
manufacturero The resolution is checked by suitable means,
for example by recording the spectrum of a polystyrene film
Impurities in gases
approximately 35 ~lm in thickness. The difference between
the absorbances at tbe absorption minimum at 2870 cm- 1 For the analysis of impurities, use a ceJl transparent to
and the absorption maÍcimum at 2849.5 cm- 1 is greater tban infrared radiation and of suitable optical path length (for
0.33. The difference between tbe absorbances at tbe example, 1-20 m). Fill tbe ceJl as prescribed under Gases.
absorption minimum at 1589 cm- 1 and tbe absorption For detection and quantification of tbe impurities, proceed as
maximum at 1583 cm- 1 is greater tban 0.08. prescribed in tbe monograph.
Verification ofthe wave-number scale The wave- Near-infrared spectrophotometry
number scale may be verified using a polystyrene film, which
has transmission minima (absorption maxima) at the wave (Ph. Eur. methad 2.2.40)
numbers (in cm- 1) shown in Table 2.2.24.-1. Near-infrared (NlR) spectrophotometry is a technique witb
wide and varied applications in pharmaceutical analysis.
The NIR spectral range extends from about 780 nm to about
2500 nm (from about 12800 cm- 1 to about 4000 cm- 1).
In sorne cases tbe most useful information is found in tbe
spectral range from about 1700 nm to about 2500 nm (from
about 6000 cm- 1 to 4000 cm- 1) . NIR spectra are dominated
by C-H, N-H, O-H and S-H overtone resonances and
combinations of fundamental vibrational modes; tbey have a
high informative character if the information is extracted by
suitable chemometric algoritbms. NIR bands are much
2014 Appendix II A V-A161
weaker than the fundamental mid-IR vibrations from which hieasure~enthiedhods

they originate. Because molar absorptivities in the NIR range Transmission mode Transmittance (7) is a measure of
are low, radiation typically penetrates several millimeters into the decrease in radiation intensity at given wavelengths when
materials, including solids. Furthermore, many materials such radiation is passed through the sample. The sample is placed
as glass are relatively transparent in this region. in the optical beam berween the source and detector.
Measurements can be made directly on in situ samples, in The arrangement is analogous to that in many conventional
addition to standard sampling and testing procedures. spectrophotometers and the result can be presented directly
Physical as well as chemical information, both qualitative and in rerms of transmittance (7) or/and absorbance (A).
quantitative, is available from NIR spectra. However, direct
comparison of the spectrum obtained with the substance 1
T
being examined with a reference spectrum of a chemical lo'
reference substance, as used in infrared absorption lo intensity of incident radiation,
spectrophotometry, is not appropriate . Suitable validated 1 intensity of transmitted radiation,
mathematical treatrnent of the data is required.
NIR spectrophotometry has a wide variety of applications for
both chemical and physical analysis, for example:
A - loglO T= loglO ( ~) = loglO ( ; ) .
chemical analysis
Diffuse reflection mode The diffuse refiection mode
- identification of active substances, excipients, dosage gives a measure of refiectance (R), the ratio of the intensity
forms, manufacturing intermedia tes, chemical raw of light refiected from the sample (1) to that refiected from a
materials and packaging materials, background or reference refiective surface (1r) ' NIR radiarion
- quantification of active substances and excipients, can penetrate a substantial distance into the sample, where it
determination of chemical values such as hydroxyl value, can be absorbed by vibrational combinations and overtone
iodine value, acid value, determination of water content, resonances of the analyte species present in the sample. Non-
determination of degree of hydroxylation, control of absorbed radiation is refiected back from the sample to rhe
solvent content, detector. NIR refiectance spectra are typically obtained by
- process control. calculating and plotting log (lIR ) versus the wavelength or
physical analysis wavenumbers.
- crystalline form and crystallinity, polymorphism, 1
pseudopolymorphism, particle size, R
Ir '
- dissolution behaviour, disintegration pattem, hardness,
intensity of light diffusively refiected from the sample,
- examination of film properties,
intensity of light refiected from the background or
- process control, for example monitoring of blending and reference refiective surface,
granulation.
Measurements in the NIR region are infiuenced by many
chemical and physical factors as described below;
reproducibility and relevance of results depend on control of Transflection mode This mode is a combination of
these factors and measurements are usually valid only for a
transmittance and refiectance. In the measurement of
defined calibration model.
transfiectance (1'*) a mirror or a diffuse refiectance surface is
Apparatus used 'to refiect the radiation transmitted through the sample
a second time and thus doubling the pathlength. Non-
NIR spectrophotometers are used for recording spectra in the absorbed radiation is refiected back from the sample to the
region of about 780 nm to about 2500 nm (about detector.
12800 cm- 1 to about 4000 cm- 1). All NIR measurements
are based on passing light through or into a sample and 1
1'*
measuring the attenuation of the emerging (transmitted, IT'
scattered or refiected) beam. Spectrophotometers for
measurement in the NIR region consist of a suitable light Ir intensity of transfiecred radiation, without sample,
1 intensity of transmitted and refiected radiation
source, a monochromator or interferometer. Common
measured with the sample,
monochromators are acousto-optical tuneable filters (AOTF),
gratings or prisms. High intensity light sources such as quartz
or tungsten lamps or similar are used. The tungsten lamp
A* lOglO ( ~* ) .
light source can be highly stabilised. Therefore many NIR
instruments have the single-beam designo Silicon, lead
S~ple preparationlpresentation
sulfide, indium arsenide, indium gallium arsenide, mercury
cadmium telluride (MCT) and deuterated triglycine sulfate Transmission mode The measurement of transmittance
are cornmonly used detector marerials. Conventional .cuvette (7) is dependent on a background transminance spectrum
sample holders, fibre-optic probes, transmission dip cells and for its calculation. A background reference can be air, an
spinning or traversing sample holders are a few common empty cell, and a solvent blank or in special cases a reference
sampling devices. The selection is based on the intended sample. The method generally applies to liquids, diluted or
application, paying particular attention to the suitability of undiluted, dispersions, solutions and solids.
the sampling system for the type of sample to be analysed. For transmittance measurements of solids, a suitable sample
Suitable data processing and evaluation units are usually part accessory is to be used. The samples are examined in a cell
of the system. of suitable pathlength (generally 0.5-4 mm), transparent ro
NIR radiation, or by immersion of a fibre optic probe of a
suitable configuration, which yields a spectrum situated in a representative of those used for calibration. If samples of
zone of transmission compatible with the specifications of the different age are to be analysed, potential differences in the
apparatus and appropriate for the intended purpose. properties must be accounted for o
Diffuse reflection mode This method generally applies to Control of instrument performance
solids. The sample is examined in a suitable device. Care
must be taken to make the measuring conditions as Use the apparatus according to the manufacturer's
reproducible as possible from one sample to another. When instructions and carry out the prescribed verification at
immersing a fibre optic probe in the sample, care must be regular intervals, according to the use of the apparatus and
taken in the positioning of the probe to ensure that it the substances to be tested.
remains stationary during the acquisition of the spectra and Verification of the wavelength scale (except for filter
that the measuring conditions are as reproducible as possible apparatus) Verify the wavelength scale employed,
from one sample to another. The reflected radiation of a generally in the region between about 780 nm and about
background reference is scanned to obtain the baseline, and 2500 nm (about 12 800 cm- I to about 4000 cm- I ) or in
then the reflectance of one or more analytical samples is the intended spectral range using one or more suitable
measured. Common reflectance references are ceramic tiles, wavelength standards which have characteristic maxima or
perfluorinated polymers and gold. Other suitable materials minima within the range of wavelengths to be used.
may be used. Only spectra measured against a background For example, methylene chloride or a mixture of rare-earth
possessing the same optical properties can be directly oxides are suitable reference materials. Take one spectrum
compared with one another. The particle size, water of with the same spectral resolution used to obtain the certified
hydration and state of solvation must be taken into value, and measure the position of at least 3 peaks
consideration. distributed over the range used. Acceptable tolerances are
Transflection mode A reflector is placed behind the ± 1 nm at 1200 nm, ± 1 nm at 1600 nm and ± 1.5 nm at
sample so as to double the pathlength. This configuration 2000 nm (± 8 cm- I at 8300 cm- I , ± 4 cm- I at
can be adopted to share the same instrument geometry with 6250 cm- I and ± 4 cm- I at 5000 cm- I ) . For the reference
reflectance and fibre optic probe systems where the source material used, apply the tolerance for the nearest wavelength
and the detector are on the same side of the sample. (wavenumber) from the aboye for each peak used. For FT
The sample is examined in a cell with a mirror or a suitable instruments, the calibration of the wavenumber scale may be
diffusive reflector, made either of metal or of an inert performed using a narrow water-vapour line at
substance (for example titanium dioxide) not absorbing in 7299.86 cm- I or a narrow line from a certified material.
the NIR region. For rare-earth oxides, NIST 1920 (a) is the most appropriate
reference .
Factors AtIecting Spectral Response Measurement in transmission mode Methylene
Sample temperature This parameter is important for ehlaride R may be used at an optical pathlength of 1.0 mm.
aqueous solutions and many liquids, where a difference of a Methylene chloride has characteristic sharp bands at
few degrees can result in substantial spectral changes. 11 55 nm, 1366 nm, 1417 nm, 1690 nm, 1838 nm,
T emperature is also an important parameter for solids and · 1894 nm, 2068 nm and 2245 nm. The bands at 1155 nm,
powders containing water. 1417 nm, 1690 nm and 2245 nm are used for calibration.
Other suitable standards may also be used.
Moisture and solvent residues Moisture and solvent
residues present in the samples will contribute significant Measurement in diffuse reflection (reflectance) mode
absorption bands in the NIR region. A mixture of dysprosium, holmium and erbium oxides
(1 + 1+ 1 by mas s) or other certified material may be used.
Sample thickness Sample thickness is a known source of
This reference material exhibits characteristic peaks at
spectral variability and must be understood and/or
1261 nm, 1681 nm and 1935 nm. If it is not possible to use
controlled. For example, in a reflection measurement the
extemal solid standards and if measurements of diffuse
sample may be "infinitely" thick, or thinner samples of
reflection are carried out in cells or if fibre optic pro bes are
constant thickness must have a stable, diffusively reflecting
used, a suspension of 1.2 g of titanium diaxide R in about
backing material of constant, and preferably high reflectivity.
4 mL of methylene ehlaride R, vigorously shaken, is used
Sample optical properties In solids, both surface and directly in the cell or pro be. The spectrum is recorded after
bulk scattering properties of samples must be taken into 2 minoTitanium dioxide has no absorption in the NIR
account. Spectra of physically, chemically or optically range. Spectra are recorded with a maximum nominal
heterogeneous samples may require sample averaging by instrument bandwidth of 10 nm at 2500 nm (16 cm- I at
increasing the beam size or examining multiple samples or 4000 cm- I ). Measurement is made of the position of at least
spinning the probe. Certain factors such as differing degree 3 peaks distributed over the range used. The acceptance
of compaction or particle size in powdered materials and tolerances are given under Verification of the wavelength
surface finish can cause significant spectral differences. scale. For the reference material used, apply the tolerance for
Polymorphism The variations in crystalline structure the nearest wavelength (wavenumber) for each peak used.
(polymorphism) influence the spectra. Hence different Verification ofthe wavelength repeatability (except for
crystalline forms as well as the amorphous form of a solid filter apparatus) Verify the wavelength repeatability using
may be distinguished from one another on the basis of their suitable standards . The standard deviation of the wavelength
NIR spectra. Where multiple crystalline forms are present, is consistent with the specifications of the instrument
care must be taken to ·ensure that the calibration standards manufacturer.
have a distribution of forms relevant to the intended
application. Verification ofphotometric linearity and response
stability Verification of photometric linearity is
Age of samples Samples may exhibit changes in their demonstrated with a set of transmission or reflection
chemical, physical or optical properties over time. Care must standards with known values of transmittance or reflectance
be taken to ensure that samples for NIR analysis are in percentage. For reflectan ce measurements, carbon-doped
2014 Appendix JI A V-A163
polyrner standard s are available . At least 4 reference Electronic raw data for the preparation of the spectral library
standards in the range of 10-90 per cent such as 10 per cent, must be archived.
20 per cent, 40 per cent and 80 per cent with respective Pre-treatment of data In many cases, and particularIy for
absorbance values of 1.0,0.7,0.4 and 0.1 are used. !fthe refiection mode spectra, sorne form of mathematical
system is used for analytes with absorbances higher than 1.0, pretreatrnent of the spectrum may be useful before the
a 2 per cent and/or 5 per cent standard is added to the set. development of a c1assification or calibration mode!. The aim
Plot the observed absorbance values against the reference can be, for example, to reduce baseline variations, to reduce
absorbance values and perform a linear regression. the impact of known variations that are interfering in the
Acceptable tolerances are l.00 ± 0.05 for the slope and subsequent mathematical models, or to compres s data before
0.00 ± 0.05 for the intercep1. use. Typical methods are multiplicative scatter correction
Spectra obtained from reftectance standards are subject to (MSC), the Kubelka-Munk transforms, spectral compression
variability due to the difference between the experimental techniques that may include windowing and noise reduction
conditions under which they were factory-calibrated and and the numerical caIculation of the first- or second-order
those under which they are subsequentIy put to use. Hence, derivative of the spectrum. Higher-order derivatives are not
the percentage reftectance values supplied with a set of recommended. In sorne cases spectra may also be
calibration standards may not be useful in the attempt to normalised, for example against the maximum absorbance,
establish an "absolute" calibration for a given instrument. the mean absorbance or the integrated absorbance area
But as long as the standards do not change chemicalIy or under the spectrum.
physicaIly and the same reference background is used as was Caution must be excercised when performing any
used to obtain the certified values, subsequent measurements mathematical transformation, as artefacts can be introduced
of the same standards under identical conditions including or essential information (important with qualification
precise sample positioning give information on long-term methods) can be 10s1. An understanding of the algorithm is
stability of the photometric response. A tolerance of required and in aIl cases the rationale for the use of
± 2 per cent is acceptable for long-term stability; this is only transform must be documented.
necessary if spectra are used without pre-treatment. Data evaluation Direct comparison of the spectrum of the
Verification of photometric noise Determine the substance under investigation is made with the individual or
photometric noise using a suitable reftectance standard, for mean reference spectra of alI substances in the database on
example white reftective ceramic tiles or reftective the basis of their mathematical correlation or other suitable
thermoplastic resins (for example, PTFE). Scan the algorithms. A set of known reference mean spectra and the
reftection standard over a suitable wavelength/wavenumber variability around this mean can be used with an algorithm
range in accordance with the manufacturer's for c1assification. There are different algorithms based on
recommendation and calculate the photometric noise as principal component analysis (PCA) combined with cluster
peak-to-peak noise. The value is approximately twice the analysis, SIMCA (soft independent modeIling by c1ass
standard deviation. The photometric noise is consistent with analogy), COMPARE functions using filters or UNEQ
the specification of the spectrophotometer. (unequal dispersed c1ass) and others used in the software of
NIR instruments or supplied as third-party software.
Identification and characterisation (qualitative The reliability of the algorithm chosen for a particular
analysis) application has to be validated. For example, correlation
Establishment of a spectral reference library Record coefficient, the sum of squared residuals or the distance
the spectra of a suitable number of batches of the substance using cluster analysis must comply with the acceptance limits
which have been fuIly tested according to established defined in the validation procedure.
specifications and which exhibit the variation typical for the
substance to be analysed (for example, manufacturer, Validation of the database
physical form, particle size). The set of spectra represents the Specificity The selectivity of the c1assification using
information for identification and characterisation that database spectra for positive identification of a given material
defines the sirnilarity border for that substance and is the and adequate discrimination against other materials in the
entry for that substance in the spectral Iibrary used to databas e is to be established during the validation procedure.
identifY the substance. The number of substances in the Acceptance thresholds are established. High thresholds
library depends on the specific application, but Iibraries that achieve a higher discriminatory power, but may cause sorne
are too big can cause sorne difficulties in discriminating errors due to the own variability of materials. Lower
between different materials and in validation. AII spectra in thresholds solve these problems, but could produce
the library used have the same:. ambiguous results. Potential chalIenges must be addressed to
- spectral range and number of data points, the spectral database. These can be materials received on site
- technique of measurement, that are similar to database members in visual appearance,
- data pre-treatrnent. chemical structure or by name. This chaIlenge must fail
identification. Independent samples of materials represented
!f sub-groups (Iibraries) are created, the aboye criteria are in the database, but not used to create it (i.e. different
applied independentIy for each group. The colIection of batches, blends) must give positive identification when
spectra in the Iibrary may be represented in different ways analysed.
defined by the mathematical technique used for
identification. These may be: Robustness The robusrness of the qualitative procedure
must also be chaIlenged to test the effect of minor changes
- aIl individual spectra representing the substance, to normal operating conditions on the analysis. There must
- a mean spectrum of each batch of substance, be no changes to pre-processing and calibration algorithm
- if necessary, a description of the variability within the parameters. Typical chaIlenges are:
substance spectra.
- effect of differences across operators on variations in distributed throughout the defined range of the calibration
environmental conditions (for example, temperature and model. Actual NIR responses that are non-linear may still be
humidity in the laboratory), valido
- effect of sample temperature, sample positioning on the Range The range of analyte reference values defines the
optical window and probe depth and compressionlpacking range of the NIR method and quantitation limits of the
of material, method. Controls must be in place to ensure that results
- replacement of instrument parts or sampling presentation outside the validated range are not accepted.
devices. Accuracy This can be determined by comparison with the
validation method or with known samples (samples of blank
Quantitative analysis and added amounts of tested substance). Accuracy can be
Establishment of a spectral reference library for a indicated by the standard error of prediction (SEP) of the
calibration model Calibration is the process of NIR method that should be in elose agreement with the data
constructing a mathematical model to relate the response of the validated method. The SEP is the standard deviation
from an analytical instrument to the properties of the of the residuals obtained from comparing the NIR results
samples. Any calibration algorithm that can be elearly with analytical reference data for the specified samples. It is
defined in an exact mathematical expression and gives demonstrated by correlation of NIR results with analytical
suitable results can be used . Record spectra of a suitable reference data, by comparison of the SEP to the reference
number of samples with known values of the content method used for validation. Altematively statistical
throughout the range to be measured (for example, content comparison methods may be used to compare NIR results
of water). Wavelengths used in the calibration model can be with reference values (paired t-test, bias evaluation) .
compared to the known bands of the analyte and those of Precision This expresses the eloseness of agreement
the matrix to verifY that the bands of the analyte of interest between a series of measurements under the prescribed
are being used by the calibration. Establish the calibration conditions. It is assessed by a minimum of 6 measurements
model with about two-thirds of the measured samples. performed according to the developed analytical method.
Compare the remaining one-third of the measured samples Precision may be considered at 2 levels, repeatability
with the database. All samples must give quantitative results (replicate measurements of the same sample with or without
within a precision interval as defined by the intended variation in sample positioning) and intermediate precision
purpose of the method. Correct quantification must be (replicate measurements by different analysts, different days
demonstrated in the presence of variations in the matrix of measurements).
within the specified range. Multiple linear regression (MLR), Robustness This ineludes the effects of variations of
partial least squares (PLS) and principal component temperature, humidity, sample handling and the inftuence of
regression (PCR) are commonly used. For PLS or PCR instrument changes.
calibrations, the coefficients or the loadings can be plotted
Outliers Outlier results from NIR measurements of a
and the regions of large coefficients compared with the
sample containing an analyte outside the calibration range
spectrum of the analyte. Raw data for the preparation of the
indicates that further testing is required. If further testing of
calibration model must be archived, without data
the sample by an appropriate analytical method gives the
pretreatment.
analyte content within the specifications, this may be
Pre-treatment of data Data pre-treatment can be defined accepted and considered to have met the specifications. Thus
as the mathematical transformation of the NIR spectral data an outlier result generated by NIR measurements of the
to enhance spectral features ami/or remove or reduce sample may still meet specifications for the analyte of
unwanted sources of variation prior to the development of interest.
the calibration model. Many suitable algorithms for data pre-
treatrnent and calibration exist. The selection is based on the Ongoing model evaluation
suitability for the intended use. Wavelength selection may NIR models validated for use are subjected to ongoing
enhance the efficiency of calibration models such as MLR performance evaluation and monitoring of validation
(for example, in partic1e-size determination) . It is useful to parameters. If discrepancies are found , corrective action will
delete certain ranges of the wavelength scale in sorne cases,
be necessary. The degree of revalidation required depends on
for example in the determination of water of hydration. the nature of the changes. Revalidation of a qualitative model
Wavelength compression may be applied to the data. will be necessary when a new material is added to the
Validation parameters Analytical performance reference library and may be necessary when changes in the
characteristics to be considered for demonstrating the physical properties of the material occur and when changes
validation of NIR methods are similar to those required for in the source of supply take place. Revalidation of a
any analytical procedure. Specific acceptance criteria for each quantitative model is required on account of changes in the
validation parameter must be consistent with the intended composition of the finished product, in the manufacturing
use of the method. process and in sources/grades of raw materials .
Specificity The relative discriminatory power and
selectivity for quantitative determination must be similar to Transfer of databases
those mentioned under Qualitative analysis. The extent of When databases are transferred to another instrument,
specificity testing is dependent on the application and the spectral range, number of data points, spectral resolution and
risks being controlled. Variations in matrix concentrations other parameters have to be taken into consideration. Further
within the operating range of the method must not affect the procedures and criteria must be applied to demonstrate that
quantitative measurement significantly. the model remains val id with the new database or new
Linearity The validation of linearity involves the instrument.
correlation of NIR results ca1culated from NIR responses
within the used algorithms to reference method results
2014 Appendix II B V-A165
Data storage Table 2.2.25.-1. - Absorption maxima ror control or wavelength

scale
Store the electronic NIR spectra, libraries and data according
to the current regulations. 241.15 nm (Ha) 404.66 nm (Hg)
Store the NIR spectra with the necessary data pre-treatrnent 253.7 nm (Hg) 435.83 nm (Hg)
for the special use (for example identification, partic1e size
287.15 nm (Ha) 486.0 nm (D~)
analysis, content of water etc.) according to the current
specifications. 302.25 nm (Hg) 486.1 nm (H~)
313.16 nm (Hg) 536.3 nm (Ha)

334.15 nm (Hg) 546.07 nm (Hg)
B. Ultraviolet and Visible Absorption 361.5 nm (Ha) 576.96 nm (Hg)
Spectrophotometry 365.48 nm (Hg) 579.07 nm (Hg)
(Ph. Eur. method 2.2.25)

Determination of absorbance The absorbance (A) of a Control of absorbance Check the absorbance using
solution is defined as the logarithm to base 10 of the suitable filters or a solution of potassium diehromate R at the
reciprocal of the transmittance (1) for monochromatic wavelengths indicated in Table 2.2.25 .-2, which gives for
radiation: each wavelength the exact value and the permitted limits of
the specific absorbance. The tabIe is based on a tolerance for
A = logIO (~ ) = loglO ( 7) the absorbance of ± 0.01.
For the control of absorbance, use soIutions of potassium
diehromate R that has been previously dried to constant mass
TIlIa , at 130 oC. For the control of absorbance at 235 nm,
10 intensity of incident monochromatic radiation, 257 nm, 313 nm and 350 nm, dissolve 57.0-63.0 mg of
1 intensity of transmitted monochromatic radiation. potassium diehromate R in 0.005 M sulfurie acid and dilute to
In the absence of other physico-chemical factors, the 1000.0 mL with the same acid. For the control of
absorbance (A) is proportional to the path length (b) through absorbance at 430 nm, dissolve 57.0-63.0 mg of potassium
which the radiation passes and to the concentration (e) of the diehromate R in 0.005 M sulfurie acid and dilute to 100.0 mL
substance in solution in accordance with the equation: with the same acid. Suitable certified reference materials may
also be used.
A = écb
TabIe 2.2.25.-2
f; = molar absorptiviry, if b is expressed in centimetres and
e in moles per litre. Wavelength Specific absorbance Maximum
The expression A:
~: cent representing the specific
(nm)
235
Al per cent
J cm
124.5
tolerance
122.9 to 126.2
absorbance of a dissolved substance refers to the absorbance
of a 10 gIL solution in a 1 cm cel! and measured at a defined 257 144.5 142.8 to 146.2
wavelength so that: 313 48.6 47.0 to 50.3
Al per cent _ 1010 350 107.3 105.6 to 109.0
1 cm - Mr
430 15.9 15.7 to 16.1
Unless otherwise prescribed, measure the absorbance at the

prescribed wavelength using a path length of 1 cm. Unless Limit of stray light Stray light may be detected at a given
otherwise prescribed, the measurements are carried out with wavelength with suitable filters or solutions: for example, the
reference to the same solvent or the same mixture of absorbance of a 12 gIL solution of potassium ehloride R in a
solvents. The absorbance of the solvent measured against air 1 cm cell increases steeply between 220 nm and 200 nm and
and at the prescribed wavelength shall not exceed 0.4 and is is greater than 2.0 at 198 nm when compared with water as
preferably less than 0.2. Plot the absorption spectrurn with compensation liquido Suitable certified reference materials
absorbance or function of absorbance as ordinate against may also be used.
wavelength or function of wavelength as abscissa. Resolution (for qualitative analysis) When prescribed in
Where a monograph gives a single value for the position of a monograph, measure the resolution of the apparatus as
an absorption maximum, it is understood that the value follows: record the spectrurn of a 0.02 per cent V/V solution
obtained may differ by not more than ± 2 nm. of toluene R in hexane R. The minimum ratio of the
Apparatus Spectrophotometers suitable for measuring in absorbance at the maximum at 269 nm to that at the
the ultraviolet and visible range of the spectrum consist of an minimum at 266 nm is stated in the monograph. Suitable
optical system capable of producing monochromatic certified reference materials may also be used.
radiation in the range of 200-800 nm and a device suitable Spectral slit-width (for quantitative anaIysis) To avoid
for measuring the absorbance. errors due to spectral slit-width, when using an instrurnent
Controlofwave1engths Verify the wavelength scale using on which the slit-width is variable at the selected wavelength,
the absorption maxima of holmium perehlorate solution R, the the slit-width must be small compared with the half-width of
line of a hydrogen or deuterium discharge lamp or the lines the absorption band but it must be as large as possible to
of a mercury vapour arc shown in Table 2.2.25 .-1. obtain a high value of lO. Therefore, a slit-width is chosen
The permitted toleran ce is ± 1 nm for the ultraviolet range such that further reduction does not result in a change in
and ± 3 nm for the visible range. Suitable certified reference absorbance reading.
materials may also be used.
V-A166 Appendix II e 2014
Cells The tolerance on the path length of the cells used is Resolution power When prescribed in a monograph,
± 0.005 cm. When filled with the same solvent, the cells record the second-order-derivative spectrum of a
intended to contain the solution 10 be examined and the 0.02 per cent VIV solution of toluene R in methanal R, using
compensation liquid must have the same transmittance. methanal R as the compensation liquid. The spectrum shows
If this is not the case, an appropriate correction must be a small negative extremum located between 2 large negative
applied. extrema at 261 nm and 268 nm, respectively, as shown in
The cells must be cleaned and handled with careo Figure 2.2.25. -1. Unless otherwise prescribed in the
monograph, the ratio A IB (see Figure 2.2 .25.-1) is not les s
Derivative spectrophotometry than 0.2.
Derivative spectropho1Ometry involves the transformation of Procedure Prepare the solution of the substance to be
absorption spectra (zero-order) into first-, second- or higher- examined, adjust the various instrument settings according to
order-derivative spectra. the manufacturer's instructions, and calculate the amount of
A firsc-order-denvacive spectrum is a plot of the gradient of the the substance 10 be determined as prescribed in the
absorption curve (rate of change of the absorbance with monograph.
wavelength, dNdA) against wavelength.
A seeond-order-denvauve spectrum is a plot of the curvature of
the absorption spectrum against wavelength (d 2NdA 2).
The second-order-derivative spectrum at any wavelength A is
C. Nuclear Magnetic Resonance
related to concentration by the following equation: Spectrometry
(Ph. Eur. mechad 2.2.33)
d 2 A~~: ce nt clb d 2 Af: cb
d..\2 x 10 = d..\2 X 10 INTRODUCTION
Nuclear magnetic resonance (NMR) spectrometry is an
el = concentration of the absorbing solute, in grams per litre . analytical method iiI particular suitable for the elucidation of
the chemical structure of organic molecules by means of
Apparatus Use a spectropho1Ometer complying with the
requirements prescribed aboye and equipped with an interpretation of their NMR spectra, arising from, for
example, IH or the X-nuclei 13C, 19F, 15N, 31 p. The spectra
analogue resistance-capacitance differentiation module or a
can be used for qualitative and quantitative purposes.
digital differentia10r or other means of producing derivative
spectra . Sorne methods of producing second-order-derivative Under suitable experimental conditions, the integrated NMR
spectra produce a wavelength shift relative 10 the zero-order intensities of the signals are directly proportional to the
spectrum and this is to be taken into account where number of nuclear spins of the molecular group responsible
applicable. for the signa!. These integral s can be used for quantitative
analysis.
2 GENERAL PRINCIPLES
Placing an ensemble of nuclei with angular momentum and a
magnetic moment in a static magnetic field (B o) causes the
nuclei to arrange themselves in different, quantum-
mechanically controlled orientations in relation 10 the axis of
the magnetic field. These orientations are different in energy.
An oscillating high-frequency magnetic field (B I ),
perpendicular to Bo, will cause transitions between these
263 orientations with net energy absorption. According 10 the
- - -- - - -- resonance condition Cúo = yBo (y = gyromagnetic ratio,
~
+ + Cúo = Larmor frequency), either the Bo magnetic fie ld or the
frequency (Cúl) ofthe BI field may be varied to achieve a
/\L\
v
/\,.A
V VV\n
spectrum (continuous wave (CW) method). Nowadays the
BI irradiation is achieved by the use of a radiofrequency (RF)
pulse (Fourier transform (FT) method). The coherent
A - radiation emitted during the retum 10 the initial state is
265
- - -- - - - observed in the form of a decay curve, called the free
induction decay (FID). Subsequent Fourier transformation
gives the spectrum in the frequency doma in, providing
B information about the molecular structure. Additional
- - - - - -- radioftequency fields may be applied during acquisition of
261
the FID signal 10 suppress scalar (through-bond) interactions
between nuclei (called 'decoupling'). One- and multi-
dimensional techniques can be applied for qualitative and
quantitative purposes, on samples in either the liquid or the
268 solid state.
Important structural information is derived from the
240 260 280 290 nm following spectroscopic features:
Figure 2.2.25.-1
2014 Appendix II e V-A167
resonance frequency kind of nuclei observed to digitisation 10 maximise sensitivity without saturating the
number of resonance signals (singlets, nurnber of chemically distinct groups
ADC. In case of observation of X-nuclei, the standard
rnultiplets) of nuclei experiment includes, if necessary, broadband IH decoupling,
chernical shift Ii (ppm) chernical nature and environrnent of i.e. irradiation of all the pro1Ons during the experiment.
the structural group observed To increase the S /N, multiple FID signals may be
intensity of resonance signals relative number of resonant nuclei per accumulated coherently and summed. Fourier transfonnation
chernically distinct group of this time-domain data gives the frequency-domain
multiplicity of coupling pattern nurnber of nuclei that are scalar spectrum.
coupled to the observed nucleus
coupling constant "J (Hz) nurnber of bonds in the coupling Parameters
I pathway,and its geornetry
The following acquisition parameters infiuence the result of
an FT experiment, and should be adjusted and controlled.
Correlations of different spectral parameters (e.g. chemical
Pulse width (l p ) . The excitation pulse is directed along
shift and coupling constant, or chemical shifts of different
the x-axis of the so-called rotating frame, its duration (or
nuclei within one molecular system) can be perfonned by
'width', lp) detennines the fiip angle (8) and thus the
homo- and hetero-nuclear two- and higher-dimensional
intensity (l) of the resonance signal:
methods. Information about the relaxation times TI and T2 ,
nuclear Overhauser effects (NO Es) and the kinetics of time-
dependent processes are also accessible from appropriate e = {' x El X Tp
(1)
experiments.
APPARATUS
My = Mo x sine
A high-resolution NMR spectrometer consists of at least the (2)
following parts:
--:. a magnet 10 deliver the constant magnetic field BO; The observed magnetisation My is maximum at 8 = 90°.
The pulse duration should be short 10 guarantee that all
- a temperature-controlled probe 10 contain the sample, to
signals in the spectral width (SW') are excited 10 a similar
deliver the radiofrequency pulse and 10 detect radiation
degree. The magnetisation decays due to relaxation
emitted by the sample;
processes.
- an electronic console to generate high-power
Dead time (t d ) The dead time is the time between the
radiofrequency pulses and to collect and digitise the FID
end of the pulse and start of the acquisition, it is necessary
signal; this unit also maintains the stability of the
for technical reasons and care should be taken as it may
instrument electronics;
infiuence signal intensities and peak phase. Rapidly decaying
- a data acquisition and processing unit (computer); signals (giving rise to broad spectral lines) are reduced in
- and may also include: intensity by more than slowly decaying signals (which give
- a continuous fiow cell for coupled liquid rise to narrow spectral lines).
chromatographic-NMR or fiow injection analysis; Acquisition time (tac) The acquisition time (tac) is related
- a system for pulsed field gradient NMR. 10 the spectral width (i.e. the whole observed region) and the
The high magnetic field is generated by a superconducting number of digital data points (DP) collected during signal
coil in a Dewar fiask filled with liquid helium. The probe acquisition.
typically contains the sample in a 5 mm-outer-diameter
sample tube or in a fiow cell, and is connected 10 the DP
t ae = 2SW
electronics cabinet by RF cables carrying lock, IH_, and (3)
X-nucleus frequencies. Additional devices for tuning and
matching the electronic circuits are essential, and sample Maximal signal intensity and signal-1O-noise ratio will be
temperature control is often used. achieved if tac ;::; 1.2/(rev1l2), where V1l2 is the full width at
The NMR spectrometer should be demonstrated 10 be half-height (jwhh), but it should be set 10 greater than
operating correctly. Appropriate tests to demonstrate this are, 5/(reV1l2) 10 minimise signal distortion.
typically, measurement of linewidths at half height for Repetition time (tr ) The spin-Iattice relaxation (TI)
defined peaks under defined acquisition conditions, signal-to- governs the time required for the spin system 10 retum 10
noise ratio s .(S/N) for standard mixtures, pulse power equilibrium after a pulse. Relaxation can be reduced by the
(measured as a 90 ~ pulse width), and pulse reproducibility. use of special reagents. For quantitative purposes, the
All instrument manufacturers publish specifications and repetition time used should be set relative 10 TI and 8 10
measurement protocols for these parameters for specific avoid saturation effects.
instrumentlprobe combinations, and compliance with these Receiver gain The analogue signal detected by the probe
specifications should be demonstrated. is amplified prior 10 digitisation and storage.
FOURIER TRANSFORM NMR (FT -NMR) The amplification, or receiver gain, should be set, either
au10matically or manually, so that the signal does not
Contemporary spectrometers generally operate according 10 overload the ADC, which causes signal dis1Ortion, but allows
the Fourier transfonn (FT) principIe: after exciting the random noise generated in the probe to be digitised
sample with a radiofrequency pulse of appropriate frequency (i.e. is non-zero) .
(v), amplitude (B I ) and duration (l p ) and a succeeding short
dead time (td) (10 enable the electronics 10 recover), the Optimisation of acquisition and processing parameters
amplified analogue FID signal is sampled during the for quantitative purposes
acquisition time (tae) and digitised with an analogue-1O-digital Besides the acquisition parameters, signal intensity is also
converter (ADC), and the results are stored in the infiuenced by several processing parameters. After collecting
spectrometer memory. The receiver output is amplified prior a sufficient number of scans, the resulting FID is Fourier
V-A168 Appendix II e 2014
transfonned. For reliable quantitative purposes the following Referencing The spectral feature most dependent on the
parameters have to be optimised. chemical environment of the atom in the molecule is the
Digital resolurlon The digital resolution is the frequency o
chemical shift, designated as and reported in parts per
separation between data points. The processed signal should million. The chemical shift between the resonance for an
have at least 5 digital points aboye half-height of the signals NMR active nuc1eus X (Üx"ample) is measured in parts per
to be integrated. To improve the digital resolution additional million as the difference between the resonance frequency of
points of zero intensity may be added to the end of the that nuc1eus (VX,sample) and that of an internal shift reference
experimental FID before transfonnation ('zero filling '). standard (VX,reference), both in hertz, divided by the basic
spectrometer operating frequency (VX,reference) , in megahertz,
Signal-to-noise ratio (SIN) This is the ratio between the
at a given Bo:
intensities (as peak height) of a specified signal in the NMR
spectrum and the random tluctuations in that signal, which
.r (VX ,sample - lIx,reference)
is usually measured in a region of the spectrum that contains UX,sample = lIX,reference (4)
no signals from the analyte. A poor signal-to-noise ratio
(S/N) limits the accuracy of peak integrations and
quantitative analyses. An S /N equal to or greater than 150: 1 By convention, the standard for exact chemical shift
allows peak integrations with a standard deviation of less referencing is the lH resonance of tetramethylsilane R (TMS),
than 1 per cent. Contemporary spectrometers have software setting O-rMS = O ppm. Fonnally, once the lH shift scale has
algorithms to report the S /N of appropriate peaks. been referenced relative to TMS, the exact frequency of any
A sufficiently high S/N can be difficult to obtain when other X resonance can be ca1culated and its chemical shift
analysing dilute solutions, and especially when detecting scale calibrated. The frequency of a (secondary) reference
nuc1ei other than lH. Methods to enhance the S/N inc1ude: standard at Ox = O ppm (VX,reference) is ca1culated from the
lH frequency ofTMS (VH,TMS) and a tabulated value ofthe
- increasing the number of accumulated scans (n), as S /N
ratio (2 X ,reference) of the isotope-specific frequency in relation
increases with Vn;
to that of lH in TMS:
- use of eXPQnentional multiplication on the FID signal
before Fourier transfonnation; the exponentional
multiplication factor should be in the order of the peak (5)
full width at half-height (fwhh);
- use of spectrometers with a higher magnetic field B o, since Reference standards at Ox = O ppm and corresponding
S /N is proportional to B o3/2; 2 X ,reference values are shown below:
- use of digital filtering to reduce noise;
- use of probes that maximise the filling factor; Water' Other
Nuc1eus :SX,rdtrenu
solvenls "'"
...... X,rderence
- use of cryoprobes that reduce thennal noise.
'H DSSb 1.00000000 TMS 1.00000000
Integrarlon region The intensity of the NMR signals is
obtained by a quasi-analogue signal integration either by a
13c DSS b
0.25144953 TMS 0.25145020
stepped-line plot or, more accurately, by separate line 15N NH, 0.10132912 CH,NO, 0.10136767
integration and digital data presentation. In liquid state, 19F CF,COOH not reported 0.94094011
CCI,F
NMR signals have Lorentzian line shape. Unless otherwise
3lp H,PO, 0.40480742 (CH,O),PO 0.40480864
specified in the monograph or when peak overlap occurs, the
(85 per cent)
same integration range, expressed as a multiple of the peak
'chemical shift depends on pH
fwhh, should be used for the monitored peak and the
reference peak. bDSS = sodium 2,2-dimethyl·2-siJapentane-5-sulfonate
Dynamic range The dynamic range of the analogue-to-
digital converter (ADC) detennines the minimum intensity In practice, X chemical shifts are referenced directly using an
line that can be observed or quantified when integrating appropriate standard. In lH and l3C NMR, internal
2 signals with the same linewidth in a spectrum. A 16-bit referencing is mainly used, where the reference is added
ADC allows identification of a signal of 0.003 per cent directly to the system under study. In 15N, 19F and 31p
intensity relative to a strong signal completely filling the NMR, external referencing is often suitable, involving sample
dynamic range of the ADC. and reference contained separately in coaxial cylindrical
NMR 01 samples in solution sample tubes .
Most NMR experiments are perfonned on dilute solutions Lock In order to prevent drifting of the spectrum over
(about 1 per cent) of the analyte in an appropriate solvent, time, a stabilising procedure, called field-frequency locking,
which can be spiked with a suitable standard for chemical is perfonned. The 2H (deuterium) signal arising from
shift calibration. deuterated solvents is used to achieve this, unless otherwise
specified in the monograph.
Solvents The solvent should be able to dissolve the analyte
without further interaction if not otherwise intended. Qualitative Analaysis
To minimise the intense solvent signals, fully deuterated The principal use for qualitative NMR spectra is as an
solvents (deuterium oxide R, deuterated chloroform R, deuterated identity test, in which the lH or l3c spectrum of a test
dimethyl sulfoxide R, deuterated acetone R, deuterated sample is compared to the spectrum of a reference sample or,
m ethanol R, etc.) should be used. The solvent atoms give less commonly, with a published reference spectrum. Spectra
signals that are easily identified by their chemical shift and of reference and test samples should be acquired using the
can be used to calibrate the chemical shift axis (secondary same procedure and operational conditions. The peaks in the
reference) . 2 spectra, or characteristic regions of the spectra, should
correspond in position, intensity and multiplicity.
In appropriate cases, mathematical comparison, such as
2014 Appendix II e V-A169
calculation of a correlation coefficient, may be appropriate. Normalisation procedure The relative proportions of
In the absence of a reference standard, an in-house reference components in a mixture, the degree of substitution in a
may be used, whose identity has been demonstrated by structuraIly modified polymer, or the amount of a
aItemative methods, or the demonstration that the NMR contaminant can be determined by comparison of the
spectrum is fuIly consistent with the reported structure for relative intensities of resonances presento
that material. The experimental method should be validated to ensure that
Quantitative Analysis there is no overlap of the relevant signals. When the
Signal intensity in the basic NMR experiment is the contaminant is of poorly defined structure or molecular mas s
integrated area under the signal curve measured. Only when (e.g. an emulsifier), addition of known amounts of that
2 signals have equal fwhh and the same multiplicity may material to the NMR tube will aIlow a calibration curve to be
signal height serve as a measure of intensity. Under constructed.
conditions of essentialIy complete relaxation between scans,
METHOD
the signal intensity (lA) is a true measure of the number (NA)
of nucIei responsible for the respective signal: Sample handling Dissolve the sample in the solvent to
which the appropriate reference material may have been
added to calibrate chemical shift, as prescribed in the
(6) monograph. For quantitative analysis, the solutions must be
free from solid particIes . Sorne quantitative analyses may
The constant Ks incIudes fundamental constants, properties require an internal standard to be incIuded, so that
of the sample and receiver parameters, and can be omitted in integrations of resonances from the test sample and the
cases where signal intensities are compared, giving the direct reference material can be compared. Appropriate references
relation between the numbers of nucIei in the 2 compared and concentrations are indicated in the specific monographs.
structure groups A and B: In other quantitative analyses, the result is obtained by
comparing the relative intensities of 2 or aIl of the
resonances that arise from the test sample. After loading the
(7) sample into a tube and capping, the sample is introduced
into the NMR magnet, the experimental parameters are
The numbers (N¡) of nucIei belonging to different structure loaded and the experiment is executed. Key experimental
groups of 1 molecule are smaIl integers. The values measured parameters are indicated in the monograph.
are rounded to the cIosest integer numbers. However, the
The measurement procedure Equilibrate the sample in
proper operation of acquisition and processing of the
the pro be, and optimise the instrument to achieve best
spectrometer is easily checked by comparing exact intensities
resonance conditions and to maximise the S /N by tuning and
within a spectrum of any suitable organic compound of
matching the probe, and make adjustrnents to maximise
known structure.
magnetic field homogeneity over the sample volume (calIed
In addition to the fact that the intensities of signals arising 'shimming'). Record, or save to computer, the parameter
from each component in a mixture are related to each other settings. An experiment may be composed of muItiple pulse-
by smalI integer numbers, the relative molar amounts of acquisition-delay sequences, and the individual FIDs are
these components can be measured by comparison of the summed in the computer memory, with random noise being
normalised intensities of resonances from different averaged out. When an appropriate S /N has been achieved,
components. The molar ratio of 2 components of a mixture the FID is stored and the frequency-domain spectrum is
is calculated according to the foIlowing equation: generated by Fourier transformation of the surnmed FIDs.
NMR in the Solid State
(8) Samples in the solid state can be analysed using NMR
spectrometers speciaIly equipped for that purpose. Certain
The determination is only valid in cases where the structure technical procedures make observable individual lines for
of the molecules for which lA and l B are determined are individual atomic sites with a valuable extension of the
known (or at least the values of N for the monitored groups). applicability of NMR to inorganic materials as weIl.
Determinations are made using either an intemal standard One technique is the rapid rotation (4-30 kHz) of the
method or a peak-normalisation procedure. powdered sample in a rotor (about 4 mm outer diameter)
Internal standard method The mass {¡nA) of an analyte incIined at an angle of 54.7° (the 'magic angle') to the
(A) can be determined if a known mass (111B) of a substance direction of the Eo magnetic field axis. This technique is
(B) with a known percentage content (PB ) is added to the named magic angle spi=ing (MAS). Another effective tool is
solution as an intensity standard. Equation (8) can be high-power decoupling and a 3 rd method is the transfer of
converted to equation (9): polarisation from easily excitable nucIei towards less-
polarisable nucIei, i.e. cross polarisation (CP).
The combination of these techniques makes available high-
(9) resolution spectra containing much information about
chemical and structural details in solid glassy, amorphous,
Here, Mi are the molecular masses. and crystalline materials of ceramic, polymeric or
mineralogical origino
The intensity standard has to be carefuIly chosen; it should
be completely soluble in the solvent used for the analyte, If NMR is applied to a solid, fuIl details of the procedure are
should produce only a smalI number of signals, and the provided in the monograph.
'monitor group' should have a signal in an empty spectral
region. A compound of high purity and with a relatively high
molecular mass is recommended for this purpose.
V-A170 Appendix lID 2014
Method
D. Atomic Spectrophotometry: Emission Use of plastic labware is recommended wherever possible.
and Absorption Operate an atomic emission spectrometer in accordance with
the manufacturer's instructions at the prescribed wavelength.
Atomic emission spectrometry
Optimise the experimental conditions (flame temperature,
(Ph. Eur. method 2.2.22) bumer adjusttnent, use of an ionic buffer, concentration of
General principIe solutions) for the specific element to be analysed and in
respect of the sample matrix. Introduce a blank solution into
Atomic emission is a process that occurs when
electromagnetic radiation is emitted by excited atoms or ions. the atomic generator and adjust the instrument reading to
In atomic emission spectrometry the sample is subjected to zero or to its blank value. Introduce the most concentrated
reference solution and adjust the sensitivity to obtain a
temperatures high enough to cause not only dissociation into
atoms, but also to cause significant amounts of collisional suitable reading.
excitation and ionisation of the sample atoms to take place. It is preferable to use concentrations which fal! within the
Once the atoms and ions are in the excited states, they can linear part of the calibration curve. If this is not possible, the
decay to lower states through thermal or radiative (emission) calibration plots may also be curved and are then to be
energy transitions and electromagnetic radiation is emitted. applied with appropriate calibration software.
An emission spectrum of an element contains several more Determinations are made by comparison with reference
lines than the corresponding absorption spectrum. solutions with known concentrations of the element to be
Atomic emission spectrometry is a technique for determining determined either by the method of direct calibration
the concentration of an element in a sample by measuring (Method 1) or the method of standard additions (Method Il).
the intensity of one of the emission lines of the atomic
vapour of the element generated from the sample. Method 1 - Direct calibration
The determination is carried out at the wavelength For routine measurements 3 reference solutions of the
corresponding to this emission lineo element to be determined and a blank are prepared and
In this chapter only atomisation in flame is dealt with. examined.
The method of inductive1y coupled plasma-atomic emission Prepare the solution of the substance to be examined (test
spectrometry (ICP-AES) is described in a different general solution) as prescribed in the monograph. Prepare not fewer
chapter. than 3 reference solutions of the element to be determined,
the concentrations of which span the expected value in the
Apparatus
test solution. For assay purposes, optimal calibration levels
This consists essential1y of: are between 0.7 and l.3 times the expected content of the
- a sample introduction and nebulisation system; element to be determined or the limit prescribed in the
- a flame to generate the atoms to be determined; monograph. For purity determination, calibration levels are
- a monochromator; between the limit of detection and 1.2 times the limit
specified for the element to be determined. Any reagents
- a detector;
used in the preparation of the test solution are added to the
- a data-acquisition unit. reference solutions and to the blank solution at the same
Oxygen, air and a combustible gas such as hydrogen, concentration.
acetylene, propane or butane may be used in flames. Introduce each of the solutions into the instrument using the
The atomisation source is critical, since it must provide same number of replica tes for each solution, to obtain a
sufficient energy to excite and atomise the atoms. The atomic steady reading.
spectra emitted from flames have the advantage of being
Calculation Prepare a calibration curve from the mean of
simpler than those emitted from other sources, the main
the readings obtained with the reference solutions by plotting
limitation being that the flames are not powerful enough to
cause emission for many elements al!owing their the means as a function of concentration. Determine the
concentration of the element in the test solution from the
determination. Acidified water is the solvent of choice for
curve obtained.
preparing test and reference solutions, although organic
solvents may also be used if precautions are taken to ensure
Method 11 - Standard additions
that the solvent do es not interfere with the stability of the
flameo Add to at least 3 similar volumetric flasks equal volumes of
the solution of the substance to be examined (test solution)
Interferences prepared as prescribed. Add to al! but 1 of the flasks
Spectral interference is reduced or eliminated by choosing an progressively larger volumes of a reference solution
appropriate emission line for measurement or by adjusting containing a known concentration of the element to be
the slit for spectral band-width. Physical interference is determined to produce a series of solutions containing
corrected by diluting the sample solution, by matching the steadily increasing concentrations of that element known to
matrix or by using the method of standard additions. give responses in the linear part of the curve, if at al!
Chemical interference is reduced by using chemical modifiers possible. Dilute the contents of each flask to volume with
or ionisation buffers. solvento
Memory effect Introduce each of the solutions into the instrument using the
The memory effect caused by deposit of analyte in the same number of replicates for each solution, to obtain a
apparatus may be limited by thoroughly rinsing between steady reading.
runs, diluting the solutions to be measured if possible and Calculation Calculate the linear equation of the graph
thus reducing their salt content, and by aspirating the using a least-squares fit, and derive from it the concentration
solutions through as swiftly as possible. of the element to be determined in the test solution.
2014 AppendixIlD V-A1?1
Validation of the method The plasma is formed by a tangential srream of support gas
Satisfactory performance of methods prescribed in through a 'torch', i.e. a system consisting of 3 concentric
monographs is verified at suitable time intervals. quartz tubes. A metal coil (the load coi!) surrounds the top
end of the torch and is connected to a radio-frequency (RF)
Linearity generator. Power (usually 700-1500 W) is applied through
Prepare and analyse not fewer than 4 reference solutions over the coil and an oscillating magnetic field corresponding to the
the calibration range and a blank solution. Perform not fewer frequency of the generator (in most cases 27 MHz, 40 MHz)
than 5 replicates. is formed. The plasma forms when the support gas is made
The calibration curve is calculated by least-square regression conductive by exposing it to an electric discharge, which
from all measured data. The regression curve, the means, the produces seed electrons and ions. Inside the induced
measured data and the confidence interval of the calibration magnetic field, the charged particles (elecrrons and ions) are
curve are plotted . The operating method is valid when: forced to flow in a closed annular path. As they meet
resistance to their flow, heating takes place producing
- the correlation coefficient is at least 0.99,
additional ionisation. The process occurs almost
- the residuals of each calibration level are randomly instantaneously, and the plasma expands to its full strength
distributed around the calibration curve. and dimensions . The radio-frequency oscillation of the power
Calculate the mean and relative standard deviation for the applied through the coil causes radio-frequency electric and
lowest and highest calibration level. magnetic fields to be set up in the area at the top of the
When the ratio of the estimated standard deviation of the torcho When a spark (produced by a Tesla tube or sorne
lowest and the highest calibration level is less than 0.5 or other seeding device) is applied to the support gas flowing
great.er than 2.0, a more precise estimation of the calibration through the torch, sorne electrons are stripped from the
curve may be obtained using weighted linear regression. Both support gas atoms. These e1ectrons are then caught up in the
linear and quadratic weighting functions are applied to the magnetic field and accelerated. Adding energy to the
data to find the most appropriate weighting function to be electrons by the use of a coil is known as inductive coupling.
employed. If the means compared to the calibration curve These high-energy electrons in tum collide with other
show a deviation from linearity, two-dimensionallinear support-gas atoms, srripping off still more electrons.
regression is used . The collisional ionisation of the support gas continues in a
chain reaction, breaking down the gas into a physical plasma
Accuracy consisting of support-gas atoms, electrons and support-gas
Verify the accuracy preferably by using a certified reference ions. The plasma is then sustained within the torch and load
material (CRM). Where this is not possible, perform a test coil as radio-frequency energy is continually transferred to it
for recovery. through the inductive coupling process.
Recovery For assay determinations a recovery of The ICP appears as an intense, very bright, plume-shaped
90 per cent to 110 per cent is to be obtained. For other plasma. At the base the plasma is toroidal, and this is
determinations, for example for trace element determination, referred to as the induction regíon (IR), i.e. the regíon in
the test is not valid if recovery is outside of the range which the inductive energy transfer from the load coil to the
80 per cent to 120 per cent at the theoretical value. plasma takes place. The sample is introduced through the
Recovery may be determined on a suitable reference solution induction regíon into the centre of the plasma.
(matrix solution) which is spiked with a known quantity of Apparatus
analyte (middle concentration of the calibration range) .
The apparatus consists essentially of the following elements:
Repeatability - sample-introduction system consisting of a peristaltic
The repeatability is not greater than 3 per cent for an assay pump delivering the solution at constant flow rate into a
and not greater than 5 per cent for an impurity test. nebuliser;
- radio-frequency (RF) generator;
Limit of quantification - plasma torch;
Verify that the limit of quantification (for example, - transfer optics focussing the image of the plasma at the
determined using the 10 (J approach) is below the value to entrance slit of the spectrometer; radial viewing is better
be measured. for difficult matrices (alkalis, organics), whereas axial
Inductively Coupled Plasma-atomic Emission . viewing gíves more intensity and better detection limits in
Spectrometry simple matrices;
(Ph. Eur. methad 2.2.57) - wavelength dispersive devices consisting of diffraction
gratings, prisms, filters or interferometers;
General principle - detectors converting radiant energy into electrical energy;
Inductively coupled plasma-atomic emission spectrometry
- data-acquisition unit.
(ICP-AES) is an atomic emission spectrometry method that
uses an inductively coupled plasma (ICP) as the excitation Interference
source. Interference is anything that causes the signal from an analyte
An ICP is a highly ionised inert gas (usually argon) with in a sample to be different from the signal for the same
equal numbers of electrons and ions sustained by a radio- concentration of that analyte in a calibration solution.
frequency (RF) field . The high temperature reached in the The well-known chemical interference that is encountered in
plasma successively desolvates, vaporises, excites - atomic flame atomic absorption specrrometry is usually weak in ICP-
emission spectrometry (AES) detection - and ionises - mass AES. In rare cases where interference occurs, it may be
spectrometry (MS) detection - atoms from the sample. necessary to increase the RF power or to reduce the inner
Detection limits are, generally, in the lower nanogram (ICP- support-gas flow to eliminate it. The interference in ICP-
MS) to microgram (ICP-AES) per litre range. AES can be of spectral origín or even the result of high
V-A172 Appendix II D 2014
concentrations of certain elements or matrix compounds. The peristaltic pumps used for ICP-AES usually deliver the
Physical interference (due to differences in viscosity and standard and sample solutions at arate of 1 mlJmin or less.
surface tension of the sample and calibration standards) can In the case of organic solvents being used, the introduction
be minimised by dilution of the sample, matrix matching, use of oxygen must be considered to avoid organic layers.
of intemal standards or through application of the method of
CHOICE OF OPERATING CONDITIONS
standard additions.
The standard operating conditions prescribed by the
Another type of interference occasionally encountered in manufacrurer are to be followed. Usually, different sets of
ICP-AES is the so-called 'easily ionised elements (EIEs) operating conditions are used for aqueous solutions and for
effect'. The EIEs are those elements that are ionised much organic solvents. Suitable operating parameters are to be
more easily, for example alkaline metals and alkaline earths. properly chosen:
In samples that contain high concentrations of EIEs (more
- wavelength selection;
than 0.1 per cent), suppression or enhancement of emission
signals is likely to occur. - suppott-gas fiow rates (ourer, intermediate and inner
tubes of the torch);
Spectral interference This may be due to other lines or
shifts in background intensity. These lines may correspond to - RFpower;
argon (observed aboye 300 nm), OH bands due to the - viewing position (radial or axial);
decomposition of water (at about 300 nm), NO bands due - pump speed;
to the interaction of the plasma with the ambient air - conditions for the detector (gainlvoltage for
(between 200 nm and 300 nm), and other elements in the photomultiplier tube detectors, others for array detectors);
sample, especially those present at high concentrations.
- integration time (time set to measure the emission
The interference falls into 4 different categories: simple
intensity at each wavelengrh) .
background sliift, sloping background shift, direct spectral
overlap, and complex background shift. Control of instrument performance
Absorption interference This arises when patt of the SYSTEM SUITABILITY
emission from an analyte is absorbed before it reaches the The following tests may be carried out with a multi-elernent
detector. This effect is observed particularly when the control solution to ensure the adequate performance of the
concentration of a strongly emitting elernent is so high that ICP-AES system:
the atoms or ions of that element that are in the lower - energy transfer (generator, torch, plasma); measurement
energy state of transition absorb significant amounts of the of the ratio Mg JI (280.270 nm)/Mg 1 (285.213 nm) may
radiation emitted by the relevant excited species. This effect, be used;
known as self-absorption, determines the upper end of the - sample transfer, by checking nebuliser efficiency and
linear working range for a given emission line. stability;
Multicomponent spectral fitting Multiple emission-line - resolution (optical system), by measuring peak widths at
determinations are commonly used to overcome problems halfheight, for example As (189 .042 nm), Mn
with spectral interferences . A better, more accurate method (257.610 nm), Cu (324.754 nrn) or Ba (455.403 nm);
for performing spectral interference corrections is to use the
- analytical performance, by calculating detection limits of
information obtained with advanced detector systems
selected elements over the wavelength range.
through multicomponent spectral fitting. This quantifies not
only the interference, but also the background contribution Validation of the method
from the matrix, thereby creating a correction formula. Satisfactory performance of methods prescribed in
Multicomponent spectral fitting utilises a multiple linear- monographs is verified at suitable time intervals.
squares model based on the analysis of pure analyte, the
LINEARITY
matrix and the blank, creating an interference-corrected
Prepare and analyse not fewer than 4 reference solutions over
mathematical mode!. This permits the determination of the
the calibration range plus a blank. Perform not fewer than 5
analyte ernission in a complex matrix with improved
replica tes.
detection limits and accuracy.
The calibration curve is calculated by least-square regression
Procedure from all measured data of the calibration test. The regression
SAMPLE PREPARATION AND SAMPLE INTRODUCTION curve, the means, the measured data and the confidence
The basic goal for the sample preparation is to ensure that interval of the calibration curve are plotted. The operating
the analyte concentration falls within the working range of method is valid when:
the instrument through dilution or preconcentration, and that - the correlation coefficient is at least 0.99;
the sample-containing solution can be nebulised in a
- the residuals of each calibration level are randomly
reproducible manner.
distribured around the calibration curve.
Several sample-introduction systems tolerate high acid
Calculate the mean and relative standard deviation for the
concentrations, but the use of sulfuric and phosphoric acids
lowest and for the highest calibration leve!.
can contribute to background emission observed in the ICP
spectra. Therefore, nitric and hydrochloric acids are When the ratio of the estimated standard deviations of the
preferable. The availability of hydrofiuoric acid-resistant (for lowest and the highest calibration level is les s than 0.5 or
example perfiuoroalkoxy polymer) sample-introduction greater than 2.0, a more precise estimation of the calibration
systems and torches also allows the use of hydrofiuoric acid. curve may be obtained using weighted linear regression. Both
In selecting a sample-introduction method, the requirements linear and quadratic weighting functions are applied to the
for sensitivity, stability, speed, sample size, corros ion data to find the most appropriate weighting function to be
resistance and resistance to c10gging have to be considered . employed.
.The use of a cross-fiow nebuliser combined with a spray
chamber and torch is suitable for most requirements.
2014 Appendix II D V-A173
If the means compared to the calibration curve show a - Flame technique

deviation from linearity, two-dimensionallinear regression is A flame atomiser is composed of a nebulisation system with a
used. pneumatic aerosol production accessory, a gas-flow regulation
ACCURACY and a burner. Fuel-oxidant mixtures are commonly used to
Verify the accuracy preferably by using a certified reference produce a range of temperatures from about 2000 K to 3000
material (CRM). Where this is not possible, perform a test K. Fuel gases inc1ude propane, hydrogen and acetylene;
for recovery. air and nitrous oxide are used as oxidants . The configuration
Recovery For assay determinations a recovery of of the bumer is adapted to the gases used and the gas flow is
90 per cent to 110 per cent is to be obtained. The test is not adjustable. Samples are nebulised, acidified water being the
valid if recovery, for example for trace-element solvent of choice for preparing test and reference solutions .
determination, is outside of the range 80 per cent to Organic solvents may also be used if precautions are taken to
120 per cent of the theoretical value. Recovery may be ensure that the solvent does not interfere with the stability of
determined on a suitable reference solution (matrix solution) the flame o
spiked with a known quantity of analyte (concentration range - Electrothermal atomisation technique
that is relevant to the samples to be determined). An electrothermal atomiser is generally composed of a
REPEAT ABILITY graphite tube furnace and an electric power source.
The repeatability is not greater than 3 per cent for an assay Electrothermal atomisation in a graphite tube furnace
and not greater than 5 per cent for an impurity test. atomises the entire sample and retains the atomic vapour in
the light path for an extended periodo This improves the
LIMIT OF QUANTIFICATION
detection limito Samples, liquid as well as solid, are
Verify that the limit of quantification (for example, introduced directly into the graphite tube fumace, which is
determined using the lOa approach) is below the value to heated in a programmed series of steps to dry the sample and
be measured. remove major matrix components by pyrolysis and to then
atomise aH of the analyte. The furnace is c1eaned using a
Atomic absorption spectrometry
final temperature higher than the atomisation temperature.
(Ph. Eur. method 2.2.23) The flow of an inert gas during the pyrolysis step in the
General principle graphite tube furnace allows a better performance of the
subsequent atomisation process.
Atomic absorption is a process that occurs when a ground
sta te-ato m absorbs electromagnetic radiation of a specific - Cold vapour and hydride technique
wavelength and is elevated to an excited state . The atoms in The atomic vapour may also be generated outside the
the ground state absorb energy at their resonant frequency spectrometer. This is notably the case for the cold-vapour
and the electromagnetic radiation is attenuated due to method for mercury or for certain hydride-forming elements
resonance absorption. The energy absorption is virtually a such as arsenic, antimony, bismuth, selenium and tino
direct function of the number of atoms presento For mercury, atoms are generated by chemical reduction
This chapter provides general information and defines the with stannous chloride or sodium borohydride and the
procedures used in element determinations by atomic atomic vapour is swept by a stream of an inert gas into a cold
absorption spectrometry, either atomisation by flame, by quartz cell mounted in the optical path of the instrumento
electrothermal vaporisation in a graphite furnace, by hydride Hydrides thus generated are swept by an inert gas into a
generation or by cold vapour technique for mercury. heated cell in which they are dissociated into atoms.
Atomic absorption spectrometry is a technique for Interferences
determining the concentration of an element in a sample by Chemical, physical, ionisation and spectral interferences are
measuring the absorption of electromagnetic radiation by the encountered in atomic absorption measurements. Chemical
atomic vapour of the element generated from the sample. interference is compensated by addition of matrix modifiers,
The determination is carried out at the wavelength of one of of releasing agents or by using high temperature produced by
the absorption (resonance) lines of the element concemed. a nitrous oxide-acetylene flame; the use of specific ionisation
The amount of radiation absorbed is, according to the buffers (for example, lanthanum and caesium) compensates
Lambert-Beer law, proportional to the element for ionisation interference; by dilution of the sample, through
concentration. the method of standard ildditions or by matrix matching,
Apparatus physical interference due to high salt content or viscosity is
eliminated. Spectral interference results from the overlapping
This consists essentially of:
of resonance lines and can be avoided by using a different
- a source of radiation; resonance lineo The use of Zeeman background correction
- a sample introduction device; also compensa tes for spectral interference and interferences
- a sample atomiser; from molecular absorption, especiaHy when using the
- a monochromator or polychromator; electrothermal atomisation technique. The use of multi-
- a detector; element hollow-cathode lamps may also cause spectral
interference. Specific or non-specific absorption is measured
- a data-acquisition unit. in a spectral range defined by the band-width selected by the
The apparatus is usually equipped with a background monochromator (0.2-2 nm).
correction system. Hollow-cathode lamps and electrodeless
Background correction
discharge lamps (EDL) are used as radiation source.
The emission of such lamps consists of a spectrum showing Scatter and background in the flame or the electrothermal
very narrow lines with half-width of about 0.002 nm of the atomisation technique increase the measured absorbance
element being determined. values. Background absorption covers a large range of
wavelengths, whereas atomic absorption takes place in a very
There are 3 types of sample atomisers:
V-A174 Appendix II D 2014
narrow wavelengtb range of about 0.005-0.02 nm. If a solid sampling technique is applied, full details of tbe
Background absorption can in principie be corrected by using procedure are provided in the monograph.
a blank solution of exactly the same composition as tbe Ensure tbat the concentrations to be determined fall
sample, but witbout tbe specific element to be determined, preferably within tbe linear part of tbe calibration curve.
altbough this method is frequently impracticable. With tbe If this is not possible, the calibration plots may also be
electrotbermal atomisation technique tbe pyrolysis curved and are then to be applied witb appropriate
temperature is to be optimised to eliminate the matrix calibration software.
decomposition products causing background absorption. Determinations are made by comparison witb reference
Background correction can also be made by using 2 different solutions with known concentrations of tbe element to be
light sources, tbe hollow-catbode lamp tbat measures tbe determined either by the metbod of direct calibration
total absorption (element + background) and a deuterium (Method I) or tbe metbod of standard additions (Metbod Il) .
lamp witb a continuum emission from which the background
absorption is measured. Background is corrected by Method 1 - Direct calibration
subtracting the deuterium lamp signal from the hollow-
For routine measurements 3 reference solutions and a blank
catbode lamp signal. This metbod is limited in tbe spectral
solution are prepared and examined.
range on account of tbe spectra emitted by a deuterium lamp
from 190-400 nm. Background can also be measured by Prepare the solution of the substance to be examined (test
taking readings at a non-absorbing line near tbe resonance solution) as prescribed in tbe monograph. Prepare not fewer
line and then subtracting tbe results from tbe measurement than 3 reference solutions of tbe element to be determined,
at the resonance line. Another metbod for the correction of the concentrations of which span the expected value in tbe
background absorption is the Zeeman effect (based on tbe test solution. For assay purposes, optimal calibration levels
Zeeman splitting of tbe absorption line in a magnetic field) . are between 0.7 and 1.3 times the expected content of tbe
This is particularly useful when tbe background absorption element to be determined or the limit prescribed in the
shows fine structure. It permits an efficient background monograph. For purity determination, calibration levels are
correction in the range of 185-900 nm. tbe limit of detection and 1.2 times the limit specified for tbe
element to be determined. Any reagents used in tbe
Choice 01 the operating conditions preparation of the test solution are added to tbe reference
After selecting the suitable wavelengtb and slit widtb for the and blank solutions at tbe same concentration.
specific element, the need for tbe following has to be Introduce each of the solutions into the instrument using tbe
ascertained: same number of replicates for each of tbe solutions to obtain
- correction for non-specific background absorption, a steady reading.
- chemical modifiers or ionisation buffers to be added to the Calculation' Prepare a calibration curve from tbe mean of
sample as well as to blank and reference solutions, the readings obtained witb the reference solutions by plotting
- dilution of tbe sample to minimise, for example, physical the means as a function of concentration. Determine the
interferences, concentration of tbe element in tbe test solution from the
- details of tbe temperature programme, preheating, drying, curve obtained.
pyrolysis, atomisation, post-atomisation witb ramp and
hold times, Method 11 - Standard additions
- inert gas flow, Add 10 at least 3 similar volumetric flasks equal volumes of
tbe solution of tbe substance to be examined (test solution)
- matrix modifiers for electrotbermal atomisation (fumace),
prepared as prescribed. Add to aIl but I of the flasks
- chemical reducing reagents for measurements of mercury progressively larger volumes of a reference solution
or other hydride-forming elements along with cold vapour containing a known concentration of tbe element to be
cell or heating cell temperature, determined to produce a series of solutions containing
- specification of fumace design (tank, L'vov platform, etc) . steadily increasing concentrations of tbat element known to
Method give responses in the linear part of the curve, if possible.
Dilute the contents of each flask to volume witb solvent.
Use of plastic labware is recommended wherever possible.
The preparation of the sample may require a dissolution, a Introduce each of tbe solutions into tbe instrument, using tbe
digestion (mostly microwave-assisted), an ignition step or a same number of replicates for each of tbe solutions, to obtain
combination thereof in order to cJear up tbe sample matrix a steady reading.
and/or to remove carbon-containing material. If operating in Calculation Calculate tbe linear equation of the graph
an open system, tbe ignition temperature should not exceed using a least-squares fit and derive from it the concentration
600 oC, due to the volatility of sorne metals, unless otberwise of the element to be determined in tbe test soJution.
stated in the monograph. Validation 01 the method
Operate an atomic absorption spectrometer in accordance Satisfactory performance of methods prescribed in
with tbe manufacturer's instructions at tbe prescribed monographs is verified at suitabJe time intervals.
wavelength. Introduce a blank solution into the atomic
generator and adjust the instrument reading so tbat it Linearity
indicates maximum transmission . The blank value may be
Prepare and analyse not fewer tban 4 reference solutions over
determined by using solvent to zero the apparatus. Introduce
the calibration range and a blank soJution. Perform not fewer
the most concentrated reference solution and adjust the
than 5 replicates.
sensitivity to obtain a maximum absorbance reading. Rinse in
order to avoid contamination and memory effects . After The calibration curve is calculated by least-square regression
completing tbe analysis, rinse witb water R or acidified water. from all measured data. The regression curve, tbe means, tbe
measured data and tbe confidence interval of the calibration
curve are plorted. The operating metbod is valid when:
2014 Appendix II F V-A175
- the correlation coefficient is at least 0.99, solution of unknown concentration and read the result on the
- the residuals of each calibration level are randomly instrumento Calculate the concentration ex of the substance in
distributed around the calibration curve. the solution to be examined, using the formula:
Calculate the mean and relative standard deviation for the
lowest and highest calibration leve!. ex ==
When the ratio of the estimated standard deviation of the
lowest and the highest calibration level is less than 0.5 or
Cx concentrarion of the solution to be examined,
greater than 2.0, a more precise estimation of the calibration
Cs concentration of the standard solution,
curve may be obtained using weighted linear regression. Both
Ix intensity of the lighr emitted by the solution to be
linear and quadratic weighting functions are applied to the
examined,
data to find the most appropriate weighting function to be
Is intensity of the light emitted by the standard solution.
employed. If the means compared to the calibration curve
show a deviation from linearity, two-dimensional linear If the intensity of the fiuorescence is not strictly proportional
regression is used. to the concentrarion, the measurement may be effected using
a calibration curve.
Accuracy
In sorne cases, measurement can be made wirh reference to a
Verify the accuracy preferably by using a certified reference fixed súllldard (for example a fiuorescent glass or a solution
material (CRM). Where this is not possible, perform a test of another fiuorescent substance). In such cases, the
for recovery. concentration of the substance to be examined must be
Recovery For assay determinations a recovery of determined using a previously drawn calibration curve under
90 per cent to 110 per cent is to be obtained. For other the same conditions.
determinations, for example, for trace element determination
the test is not valid if recovery is outside of the range
80 per cent to 120 per cent at the theoretical value.
Recovery may be determined on a suitable reference solution F. X-Ray Fluorescence Spectrometry 1
(matrix solution) which is spiked with a known quantity of
(Ph. Eur. methad 2.2.37)
analyte (middle concentration of the calibration range).
Wavelength dispersive X-ray fiuorescence spectrometry is a
Repeatability procedure that uses the measurement of the intensity of the
fiuorescent radiation emitted by an element having an atomic
The repeatability is not greater than 3 per cent for an assay
number between 11 and 92 excitedby a continuous primary
and not greater than 5 per cent for an impurity test.
X-ray radiation. The intensity of the fiuorescence produced
Limit of quantification by a given element depends on the concentration of this
element in the sample but also on the absorption by the
Verify that the limit of quantification (for example,
matrix of the incident and fiúorescent radiation. At trace
determined using the IOcr approach) is below the value to
levels, where the calibration curve is linear, the inrensity of
be measured.
the fiuorescent radiation emitted by an element in a given
matrix, at a given wavelength, is proportional to the
concentration of this element and inversely proportional to
E. Fluorescence Spectrophotometry the mass absorption coefficient of the matrix ar this
wavelength .
[Fluorimetry] Method Set and use the instrument in accordance with the
(Ph. Eur. methad 2.2.21) instructions given by the manufacturer. Liquid samples are
Fluorimetry is a procedure which uses the measurement of placed directly in the instrument; solid samples are first
the intensity of the fiuorescent light emitted by the substance compressed into pellets, sometimes after mixing wirh a
to be examined in relation to that emitted by a given suitable binder.
standard. To determine the concentration of an element in a sample, it
Method Dissolve the substance to be examined in the is necessary to measure the net impulse rate produced by one
solvent or mixrure of solvents prescribed in the monograph, or several stanqard preparations containing known amounts
transfer the solution to the cell or the tube of the fiuorimeter of this element in given matrices and to calculate or measure
and illuminate it with an excitant light beam of the the mass absorption coefficient of the matrix of the sample to
wavelength prescribed in the monograph and as near as be analysed.
possible monochromatic. Calibration From a calibration solution or a series of
Measure the intensity of the emitted light at an angle of 90° dilutions of the element to be analysed in various matrices,
to the excitant beam, after passing it through a filter which determine the slope of the calibration curve b a from the
transmits predominantly light of the wavelength of the following equation:
fiuorescence. Other types of apparatus may be used provided
that the results obtained are identica!.
bo _1_ = Ig
For quantitative determinations, first introduce into the MM e
apparatus the solvent or mixture of solvents used to dissolve
the substance to be examined and set the instrument to zero.
Introduce the standard solution and adjustthe sensitivity of J G. Anderma1111 & M. W Kemp, Analyilcal Chemistly 301306 (1958).
the instrument so that the reading is greater than 50. If the z.H. Kalman & L. Heller, Analyucal Chemisl1Y 34946 (1962). R.e.
second adjustment is made by altering the width of the slirs, Reynolds, Jr. , The American Mineralogisl 46 ] 133 (1963). R. O. Mül/el;
a new zero setting must be made and the intensity of the Specrrochimica A cta 20 143 (1964). R. O. Mül!er, Spea1'Ochemische
standard must be measured again. Finally introduce the Analyse mil Romgenfiuoreszenz, R. Oldenburg Miinchen-Wien (1967) .
V-A176 Appendix II G 2014
flM absorption coefficient of the matrix M, calculated or Introduction of the sample

measured, The very first step of an analysis is the introduction of the
Ir! net impulse rate, sample into the apparatus without overly disturbing the
e concentraúon of the element to be assayed in the vacuum. In a common method, called direct liquid
standard preparaúon. introductwn, the sample is placed on the end of a cylindrical
Mass absorption coefficent 01 the matrix 01 the sample rod (in a quartz crucible, on a filament or on a metal
If the empirical formula of the sample to be analysed is surface). This rod is introduced into the spectrometer after
known, calculate its mass absorption coefficient from the passing through a vacuum lock where a primary intermediate
known elemental composition and the tabulated elemental vacuum is maintained between atmospheric pressure and the
mass absorption coefficients. If the elemental composiúon is secondary vacuum of the apparatus.
unknown, determine the mass absorption coefficient of the Other introduction systems allow the components of a
sample matrix by measuring the intensity of the scattered mixture to be analysed as they are separated by an
X-radiation Iv (Compton scattering) from the following appropriate apparatus connected to the mas s spectrometer.
equation: Gas chromatography/mass spectrometry The use of
suitable columns (capillary or semi-capillary) allows the end
of the column to be introduced directly into the source of
- 1- = a+ blv the apparatus without using a separator.
I1MP
Liquid chromatography/mass spectrometry This
llMP = mas s absorpúon coefficient of the sample, combination is particularly useful for the analysis of polar
Iu scattered X-radiaúon. compounds, which are insufficiently volaúle or too heat-labile
to be analysed by gas chromatography coupled with mass
Deterrnination 01 the net pulse rate 01 the elernent to be spectrometry. This method is complicated by the difficulty of
deterrnined in the sample Calculate the net impulse rate obtaining ions in the gas phase from a liquid phase, which
11fp of the e1ement to be determined from the measured requires very special interfaces such as:
intensity of the fiuorescence line and the intensity of the
background linees), allowing for any tube contaminants - direct liquid introduction: the mobile phase is nebulised, and
presento the solvent is evaporated in front of the ion source of the
apparatus,
Calculation 01 the trace content If the concentraúon of
the element is in the linear part of the calibraúon curve, it - particle-beam inteiface: the mobile phase, which may fiow
can be calculated using the following equation: at arate ofup to 0.6 mUmin, is nebulised in a
desolvation chamber such that only the analytes, in
neutral form, reach the ion source of the apparatus; this
technique is used for compounds of relative1y low polarity
with molecular mas ses of les s than 1000 Da,
- moving-belt inteiface: the mobile phase, which may fiow at
f dilution factor. arate of up to 1 mUmin, is applied to the surface of a
moving belt; after the solvent evaporates, the components
to be analysed are successively carried to the ion source of
the ·apparatus where they are ionised; this technique is
G. Mass Spectrometry rather poorly suited to very polar or heat-labile
compounds .
(Ph. Eur. method 2.2.43) Other types of coupling (electrospray, thermospray,
Mass spectrometry is based on the direct measurement of the atmospheric-pressure chemical ionisation) are considered to
ratio of the mass to the numqer of positive or negative be ionisation techniques in their own right and are described
elementary charges of ions (miz) in the gas phase obtained in the section on modes of ionisaúon.
from the substance to be analysed. This ratio is expressed in
atomic mas s units (1 a.m.u. = one twelfth the mass of 12C) Supercritical fluid chromatography/mass spectrometry
or in daltons (1 Da = the mass of the hydrogen atom). The mobile phase, usually consisting of supercritical carbon
dioxide enters the gas state after passing a heated restrictor
The ion s, produced in the ion source of the apparatus, are between the column and the ion source.
accelerated and then separated by the analyser before
Capillary electrophoresis/mass spectrometry The
reaching the detector. AlI of these operations take place in a
eluent is introduced into me ion source, in sorne cases after
chamber where a pumping system maintains a vacuum of
10- 3 to 10- 6 Pa. adding another solvent so that fiow rates of the order of a
few mierolitres per minute can be attained. This technique is
The resulting spectrum shows the relative abundan ce of the limited by the small quantities of sample introduced and the
various ionie species present as a function of miz. The signal need to use volatile buffers.
corresponding to an ion will be represented by several peaks
corresponding to the statistical distribution of the various Modes of ionisation
isotopes of that ion. This pattern is called the isotopic profile
Electron impact The sample, in the gas sta te, is ionised
and (at least for small molecules) the peak representing the
by a beam of e1ectrons whose energy (usually 70 eV) is
most abundant isotopes for each atom is called the
greater than the ionisation energy of the sample. In addition
monoisotopic peak.
to the molecular ion M+, fragments characteristic of the
Informaúon obtained in mass spectrometry is essentially molecular structure are observed. This technique is limited
qualitative (determination of the molecular mass, information mainly by the need to vaporise the sample. This makes it
on the structure from the fragments observed) or quantitative unsuited to polar, heat-labile or high molecular mas s
(using internal or external standards) with limits of detection compounds. Electron impact is compatible with the coupling
ranging from the picomole to the femtomole.
2014 Appendix II G V-A177
of gas chromatography to mas s spectrometry and sometimes Thermospray The sample, in the mobile phase consisting
with the use of liquid chromatography. of water and organic modifiers and containing a volatile
Chemical ionisation This type of ionisation involves a electrolyte (generally ammonium acetate) is introduced in
reagent gas such as methane, ammonia, nitro gen oxide, nebulised form after having passed through a metal capillary
nitro gen dioxide or oxygen. The spectrum is characterised by tube at controlled temperature. Acceptable flow rates are of
ion s of the (M + Ht or (M - H)- types, or adduct ions the order of 1 mlJmin to 2 mUmin. The ions of the
formed from the analyte and the gas used. Fewer fragments electrolyte ionise the compounds to be analysed. This
are produced than with electron impacto A variant of this ionisation process may be replaced or enhanced by an
technique is used when the substance is heat-Iabile: the electrical discharge of about 800 volts, notably when the
sample, applied to a filament, is very rapidly vaporised by the solvents are entirely organic. This technique is compatible
Joule-Thomson effect (desorption chemical ionisabon). with the use of liquid chromatography coupled with mas s
Fast-atom bombardment (FAB) or fast-ion spectrometry.
bombardment ionisation (liquid secondary-ion mass
Analysers
spectrometry LSIMS) The sample, dissolved in a
viscous matrix such as glycerol, is applied to a metal surface Differences in the performance of analysers depend mainly
and ionised by a beam of neutral atoms such as argon or on two parameters:
xenon or high-kinetic-energy caesium ions. Ions of the (M + - the range over which miz ratios can be measured, ie, the
Ht or (M - H)- types or adduct ions formed from the mass range,
marrix or the sample are produced. This type of ionisation, - their resolving power characterised by the ability to separate
well suited to polar and heat-Iabile compounds, allows two ions of equal intensity with miz ratio s differing by
molecular masses of up to 10 000 Da to be obtained. L'lM, and whose overlap is expressed as a given percentage
The technique can be combined with liquid chromatography of valley deftnition; for example, a resolving power
by adding 1 per cent to 2 per cent of glycerol to the mobile (MIL'lM) of 1000 with 10 per cent valley definition allows
phase; however, the flow rates must be very low (a few the separation of miz ratios of 1000 and 1001 with the
microlitres per minute) . These ionisation techniques also intensity retuming to 10 per cent above baseline.
allow thin-Iayer chromatography plates to be analysed by However, the resolving power may in sorne cases (time-of-
applying a thin layer of matrix to the surface of these plates. flight analysers, quadrupoles, ion-trap analysers) be
Field desorption and field ionisation and field defined as the ratio between the molecular mas s and peak
ionisation The sample is vaporised near a tungsten width at half height (50 per cent valley definition) .
filament covered with microneedles (jield ionisation) or Magnetic and electrostatic analysers The ions produced
applied to this filament (jield desorption) . A voltage of about in the ion source are accelerated by a voltage V, and focused
10 kV, applied between this filament and a counter- towards a magnetic analyser (magnetic field B) or an
electro de, ionises the sample. These two techniques mainly electrostatic analyser (electrostatic field E), depending on the
produce molecular ions M+, and (M + Ht ions and are configuration of the instrumento They follow a trajectory of
used for low polarity andlor heat-Iabile compounds. radius r according to Laplace's law:
Matrix-assisted Iaser desorption ionisation (MALDI)
The sample, in a suitable matrix and deposited on a metal m
support, is ionised by a pulsed laser beam whose wavelength - =--
z 2V
may range from UV to IR (impulses lasting from a
picosecond to a few nanoseconds) . This mode of ionisation
Two types of scans can be used to collect and measure the
plays an essential role in the analysis of very high molecular
various ions produced by the ion source: a scan of B holding
mas s compounds (more than 100 000 Da) but is limited to
V fixed or a scan of V with constant B. The magnetic
time-of flight analysers (see below).
analyser is usually followed by an electric sector that acts as a
Electrospray This mode of ionisation is carried out at kinetic energy filter and allows the resolving power of the
atmospheric pressure. The samples, in solution, are instrument to be increased appreciably. The maximum
introduced into the source through a capillary tube, the end resolving power of such an instrument (double sector) ranges
of which has a potential of the order of 5 kV. A gas can be from 10 000 to 150 000 and in most cases allows the value
used to facilitate nebulisation. Desolvation of the resulting of miz ratios to be calculated accurately enough to determine
microdroplets produces singly or multiply charged ions in the the elemental composition of the corresponding ions.
gas phase. The flow rates vary from a few micro litres per For monocharged ions, the mass range is from 2000 Da to
minute to 1 mUmin. This technique is suited to polar 15 000 Da. Sorne ions may decompose spontaneously
compounds and to the investigation of biomolecules with (metastable transitions) or by colliding with a gas (collision-
molecular mas ses of up to 100 000 Da. It can be coupled to activated dissociation (CAD» in field-free regions between
liquid chromatography or capillaty electrophoresis. the ion source and the detector. Examination of these
Atmospheric-pressure chemical ionisation (APCI) decompositions is very useful for the determination of the
Ionisation is carried out at atmospheric pressure by the structure as well as the characterisation of a specific
action of an electro de maintained at a potential of several compound in a mixture and involves tandem mas s
kilóvolts and placed in the path of the mobile phase, which spectrometry. There are many such techniques depending on
is nebulised both by thermal effects and by the use of a the region where these decompositions occur:
stream of nitro gen. The resulting ions carry a single charge - daughter-ion mode (determination of the decomposition
and are of the (M + Ht type in the positive mode and of ions of a given parent ion): BIE = constant, MIKES
the (M - H r type in the negative mode. The high fiow (Mass-analysed Ion Kinetic Energy Spectroscopy),
rates that can be used with this mode of ionisation (up to
- parent-ion mode (determination of all ions which by
2 mUmin) make this an ideal technique for coupling to
decomposition give an ion with a specific miz ratio):
liquid chromatography.
B 21E = constant,
V-A178 Appendix II G 2014
- neutral-loss mode (detection of all the ions that lose the Complete spectrum mode The entire signal obtained
same fragrnent) : over a chosen mass range is recorded. The spectrum
BIE( 1 - EIEo) 1/2 = constant, where Eo is the basic represents the relative intensity of the different ionic species
voltage of the electric sector. present as a function of miz. The results are essentially
qualitative. The use of spectral reference libraries for more
Quadrupoles The analyser consists of four parallel metal
rapid identification is possible.
rods, which are cylindrical or hyperbolic in cross-section.
They are arranged symmetrically with respect to the Fragmentometric mode (Selected-ion monitoring)
trajectory of the ions; the pairs diagonally opposed about the The acquired signal is limited ro one (single-ion monitoring
axis of symmetry of rods are connected electrically. (SIM» or several (multiple-ion monitoring (MlM» ions
The potentials ro the two pairs of rods are opposed. They characteristic of the substance to be analysed. The lirnit of
are the resultant of a constant component and an alternating detection can be considerably reduced in this mode.
component. The ions produced at the ion source are Quantitative or semiquantitative tests can be carried out
transmitted and separated by varying the voltages applied ro using external or internal standards (for example, deuterated
the rods so that the ratio of continuous voltage to alternating standards). Such tests ca=ot be carried out with time-of-
voltage remains constant. The quadrupoles usually have a fiight analysers.
mass range of 1 a.m.u. to 2000 a.m. u., but sorne may range Fragmentometric double mass spectrometry mode
up to 4000 a.m.u. Although they have a lower resolving (multiple reaction monitoring (MRM» The
power than magnetic sector analysers, they nevertheless allow unimolecular or bimolecular decomposition of a chosen
the monoisotopic profile of single charged ions to be precursor ion characteristic of the substance to be analysed is
obtained for the entire mass range. It is possible to obtain followed specifically. The selectivity and the highly specific
spectra using three quadrupoles arranged in series, Q¡, Q2, nature of this mode of acquisition provide excellent
Q3 (Q2 serves as a collision cell and is not really an analyser; sensitivity levels and make it the most appropriate for
the most commonly used collision gas is argon) . quantitative studies using suitable internal standards (for
The most common types of scans are the following: example, deuterated standards). This type of analysis can be
perforrned only on apparatus fitted with three quadrupoles in
- daughter-ion mode: Q¡ selects an miz ion whose fragrnents
series, ion-trap analysers or cyclotron-resonance analysers.
obtained by collision in Q2 are analysed by Q3,
- parent-ion mode: Q3 filters only a specific miz ratio, while Calibration
Q¡ scans a given mass range. Only the ions decomposing Calibration allows the corresponding miz value ro be
to give the ion selected by Q3 are detected, attributed to the detected signa!. As a general rule, this is
- neutral loss mode: Q¡ and Q3 scan a cenain mass range but done using a reference substance. This calibration may be
at an offset corresponding to the los s of a fragrnent external (acquisition file separate from the analysis) or
characteristic of a product or family of compounds . internal (the reference substance(s) are mixed with the
Ir is also possible to obtain spectra by combining quadrupole substance to be examined and appear on the same
analysers with magnetic or electrostatic sector instruments; acquisition file). The number of ions or points required for
such instruments are called hybn'd mass spectrometers. reliable calibration depends on the type of analyser and on
Ion-trap anaIyser The principie is the same as for a the desired accuracy of the measurement, for example, in the
quadrupole, this time with the electric fields in three case of a magnetic analyser where the miz ratio varies
dimensions. This type of analyser allows product-ion spectra exponentially with the value of the magnetic field, there
over several generations (MS") to be obtained. should be as many points as possible.
Ion-cyclotron resonance analysers lons produced in a Signa! detection and data processing
cell and subjected to a uniforrn, intense magnetic field move
Ions separated by an analyser are convened into electric
in circular orbits at frequencies which can be directly
signals by a detection system such as a phoromultiplier or an
correlated to their miz ratio by applying a Fourier transform
electron multiplier. These signals are amplified before being
algorithm. This phenomenon is called ion-cyclotron
re-converted into digital signals for data processing, allowing
resonance. Analysers of this type consist of superconducting
various functions such as calibration, reconstruction of
magnets and are capable of very high resolving power (up ro
spectra, automatic quantification, archiving, creation or use
1000 000 and more) as well as MS" spectra. However, very
of libraries of mass spectra. The various physical parameters
low pressures are required (of the order of 10- 7 Pa).
required for the functioning of the apparatus as a whole are
Time-of-flight analysers The ions produced at the ion controlled by computer.
source are accelerated at a voltage V of 10 kV to 20 kV.
They pass through the analyser, consisting of a field-free
tube, 25 cm ro 1.5 m long, generally called a flight tube.
The time (t) for an ion to travel ro the detector is 1. Inductively Coupled Plasma-mass
proponional to the square root of the miz ratio. Theoretically Spectrometry
the mas s range of such an analyser is infinite. In practice, it
is limited by the ionisation or desorption method. Time-of- (Ph. Eur. method 2.2.58)
fiight analysers are mainly used for high molecular mass Inductively coupled plasma-mass spectrometry (ICP-MS) is a
compounds (up to several hundred thousand daltons). This mass spectrometry method that uses an inductively coupled
technique is very sensitive (a few picomoles of product are plasma (ICP) as the ionisation source. The basic principies of
sufficient) . The accuracy of the measurements and the ICP formation are described in chapter 2.2.57 on inductively
resolving power of such instruments may be improved coupled plasma-aromic emission spectrometry (ICP-AES) .
considerably by using an electrostatic mirror (refiectron). ICP-MS utilises the ability of the ICP to genera te charged
ions from the element species within a sample. These ions
Signa! acquisition are then directed into a mas s spectrometer, which separates
There are essentially three possible modes. them according to their mass-to-charge ratio (miz). Most
2014 Appendix JI G V-A179
mass spectrometers have a quadrupole system or a magnetic introduction method, the requirements for sensitivity,
sector. Ions are transported from the plasma through 2 eones stability, speed, sample size, corrosion resistance and
(sampler and skimmer eones, forming the interface region) to resistance to clogging have to be considered. The use of a
the ion optics. The ion optics consist of an electrostatic lens, cross-fiow nebuliser combined with a spray chamber and
which takes ions from an area at atmospheric pressure to the torch is suitable for most requirements. The peristaltic
mass filter at a vacuum of 10-8 Pa or less, maintained with a pumps usually deliver the standard and sample solutions at a
turbomolecular pump . After their filtration, ions of the rate of 20-1000 ¡.¡lJmin.
selected mass/charge ratio are directed to a detector (channel In the case of organic solvents being used, the introduction
electromultiplier, Faraday cup, dynodes), where ion currents of oxygen must be considered to avoid organic layers.
are converted into electrical signals. The element is
quantified according to the number of ions arriving and Choice oloperating conditions
generating electrical pulses per unit time. The standard operating conditions prescribed by the
manufacturer are to be followed. Usually, different sets of
The sample-introduction system and data-handling
operating conditions are used for aqueous solutions and for
techniques of an ICP-AES system are also used in ICP-MS.
organic solvents. Suitable operating parameters are to be
Apparatus properly chosen:
The apparatus consists essentially of the following elements: - selection of eones (material of sampler and skimmer);
- sample-introduction system, consisting of a peristaltic - support-gas fiow rates (outer, intermediate and inner
pump delivering the solution at constant fiow rate into a tubes of the torch);
nebuliser; - RFpower;
- radio-frequency (RF) generator; - pump speed;
- plasma torch; - selection of one or more iso topes of the element to be
- interface region including eones to transport ions to the measured (mass) .
ion optics;
Isotope se1ection
- mass spectrometer;
- detector; Isotope selection is made using several criteria. The most
abundant isotope for a given element is selected to obtain
- data-acquisition unit.
maximum sensitivity. Furthermore, an isotope with the least
Interference interference from other species in the sample matrix and
from the support gas should be selected. Information about
Mass interference is the major problem, for example by
isobaric interferences and interferences from polyatomic ions
isobaric species that significantly overlap the mass signal of
of various types, for example hydrides, oxides, chlorides, etc.,
the ions of interest, especially in the central part of the mass
is usually available in the software of ICP-MS instrument
range (for example 40-80 a.m .u.). The combination of
manufacturers.
atomic ions leads to polyatomic or molecular interferences
(i.e. 40Ar160 with 56Fe or 4°Ar4°Ar with 80Se). Matrix
Control of instrument perfonnance
interference may also occur with sorne analytes. Sorne
samples have an impact on droplet formation or on the System suitability
ionisation temperature in the plasma. These phenomena may - Tuning of the instrument allows to monitor and adjust the
lead to the suppression of analyte signals. Physical measurement before running samples. ICP-MS mas s
interference is to be circumvented by using the method of accuracy is checked with a tuning solution containing
internal standardisation or by standard addition. The element several isotopes covering the whole range of mas ses, for
used as internal standard depends on the element to be example 9Be, 59CO, 89y, ll5In, l40Ce and 209Bi.
measured: 59CO and Jl5In, for example, can be used as - Sensitivity and short- and long-term stability are recorded.
internal standards. The instrument parameters (plasma condition, ion lenses
The prime characteristic of an ICP-MS instrument is its and quadrupole parameter) are to be optimised to obtain
resolution, i.e. the efficiency of separation of 2 close masses. the highest possible number of counts .
Quadrupole instruments are, from this point of view, inferior - Tuning for resolution and mass axis is to be done with a
to magnetic-sector spectrometers. solution of Li, Y and TI to ensure an acceptable response
over a wide range of masses.
Procedure
- Evaluation of the efficiency of the plasma to decompose
Sample preparations and sample introduction oxides has to be performed in order to minimise these
The sample preparation usually involves a step of digestion of interferences. The ratio Ce/CeO andlor BalBaO is a good
the matrix by a suitable method, for example in a microwave indicator, and a levelless than about 3 per cent is
oven. Furthermore, it is important to ensure that the analyte required.
concentration falls within the working range of the - Reduction of the formation of double-charged ions is
instrument through dilution or preconcentration, and that the made with Ba and Ce. The ratio of the signal for double-
sample-containing solution can be nebulised in a charged ions to the assigned element should be les s than
reproducible manner. 2 per cent.
Several sample-introduction systems tolerate high acid - Long-term stability is checked by running a standard first
concentrations, but the use of sulfuric and phosphoric acids and at the end of the sample sequence, controlling
can con tribute to background emission. Therefore, nitric and whether salt deposits on the eones have reduced the signal
hydrochloric acids are preferable. The availability of throughout the runo
hydrofiuoric acid-resistant (for example perfiuoroalkoxy
polymer) sample-introduction systems and torches also allows
the use of hydrofiuoric acid. In se1ecting a sample-
V-A180 Appendix II H 2014
Validation of the method multiple bond s) and less sensitive to polar bonds. Hence,
Satisfacrory perfonnance of methods prescribed in water, which has a strong infrared absorption spectrum, is a
monographs is verified at suitable time intervals. weak Raman scatterer and is thus well suited as a solvent for
Raman spectrometry.
Linearity
Apparatus Spectrometers for recording Raman spectra
Prepare and analyse not fewer than 4 reference solutions over typically consist of the following components:
the calibration range plus a blank. Perfonn not fewer than 5
- a monochromatic light source, typically a laser, with a
replicates.
wavelength in the ultraviolet, visible or near-infrared
The calibration curve is calculated by least-square regression region,
from all measured data of the calibration test. The regression
- suitable optics (lens, mirrors or optical-fibre assembly)
curve, the means, the measured data and the confidence
which directs the irradiating light ro and collects the
interval of the calibration curve are plotted. The operating
scattered light from the sample,
method is valid when:
- an optical device (monochromator or filter) that transmits
- the correlation coefficient is at least 0.99;
the frequency-shifted Raman scattering and prevents the
- the residual s of each calibration level are randomly intense incident frequency (Rayleigh scattering) from
distributed around the calibration curve. reaching the detector,
Calculate the mean and relative standard deviation for the - a dispersing device (grating or prism monochromator)
lowest and for the highest calibration leve!. combined with wavelength-selecting slits and a detector
When the ratio of the estimated standard deviations of the (usuallya photomultiplier tube),
lowest and the highest calibration level is less than 0.5 or or:
greater than 2.0, a more precise estimation of the calibration
- a dispersing device (grating or prism) combined with a
curve may be obtained using weighted linear regression. Both
multichannel detector (usually a charge-coupled device
linear and quadratic weighting functions are applied to the
(CCD»,
data ro find the most appropriate weighting function ro be
employed. or:
If the means compared to the calibration curve show a - an interferometer with a detector that records the intensity
deviation from linearity, two-dimensional linear regression is of the scattered light over time, and a data-handling
used. device that converts the data to the frequency or
wavenumber domain by a Fourier-transfonn calculation.
Accuracy
Verify the accuracy preferably by using a certified reference Preparation of the sample
material (CRM). Where this is not possible, perfonn a test Raman spectra can be obtained from solids, liquids and gases
for recovery. either directly, or in glass containers or tubes, generally
Recovery For assay detenninations a recovery of without prior sample preparation or dilution.
90 per cent ro 110 per cent is ro be obtained. The test is not A major limitation of Raman spectrometry is that impurities
valid if recovery, for example for trace-element may cause fluorescence that interferes with the detection of
detennination, is outside the range 80 per cent ro the much weaker Raman signa!. Fluorescence may be
120 per cent of the theoretical value. Recovery may be avoided by choosing a laser source with a longer wavelength,
detennined on a suitable reference solution (matrix solution) for example in the near infrared, as the exciting lineo
spiked with a known quantity of analyte (concentration range The intensity of certain Raman lines may be enhanced in a
that is relevant to the samples to be detennined). number of ways, for instance in Resonance Raman (RR) and
Repeatability by Surface Enhanced Raman Spectrometry (SERS).
The repeatability is not greater than 3 per cent for an assay Due to the narrow focus of the irradiating laser beam, the
and not greater than 5 per cent for an impurity test. spectrum is typically obtained from only a few microlitres of
sample. Hence, sample inhomogeneities must be considered,
Limit of quantification
unless the sample volume is increased, for example by
Verify that the limit of quantification (for example, rotation of the sample.
detennined using the 10 (J approach) is below the value to
be measured. Identification and quantitation using reference
substances
Prepare the substance to be examined and the reference
substance by the same procedure and record the spectra
H. Raman Spectrometry under the same operational conditions. The maxima in the
(Ph. Eur. methad 2.2.48) spectrum obtained with the substance to be examined
Raman spectrometry (inelastic light scattering) is a light- correspond in position and relative intensity to those in the
scattering process in which the specimen under examination spectrum obtained with the reference substance (CRS).
is irradiated with intense monochromatic light (usually laser When the spectra recorded in the solid state show differences
light) and the light scattered from the specimen is analysed in the positions of the maxima, treat the substance ro be
for frequency shifts. examined and the reference substance in the same manner so
Raman spectrometry is complementary ro infrared that they crystallise or are produced in the same form, or
spectrometry in the sense that the two techniques both probe proceed as described in the monograph, then record the
the molecular vibrations in a materia!. However, Raman and spectra.
infrared spectrometry have different relative sensitivities for While Beer-Lambert's law is not valid for Raman
different functional groups. Raman spectrometry is spectrometry, Raman intensity is directly proportional to the
particularly sensitive to non-polar bonds (e.g. C-C single or concentration of rhe scattering species. As for other
2014 Appendix JI J V-A181
spectroscopic techniques, quantitation can be performed - the intensity of the irradiating light,
using known amounts or concentrations of reference - differences in instrument response,
substances. Owing to the small spatial resolution of the - differences in focus and geometry at sample,
technique, care must be taken to ensure representative
samples of standards and unknowns, for example by making - differences in packing density for solid samples.
sure that they are in the same physical state or by using an Appropriate acceptance criteria will vary with the application
intemal standard for liquid samples. but a day-to-day variation of ± 10 per cent in relative band
intensities is achievable in most cases.
Identification and quantitation using spectral Establishment of a spectral reference library Record
librarles and statistical methods for c1assification the spectra of a suitable number of materials which have
and calibration been fully tested (e.g. as prescribed in a monograph) and
Control of instrument performance U se the apparatus which exhibit the variation (manufacturer, batch, crystal
according to the manufacturer's instructions and carry out modification, particle size, etc.) typical of the material to be
the prescribed calibrations and system performance tests at analysed. The set of spectra represents the information that
regular intervals, depending on the use of the apparatus and defines the similarity border or quantitative limits, which
the substances to be examined. When using Raman may be used, e.g. to identify the substance or control the
spectrometry for quantitative determinations, or when setting amount formed in a manufacturing process. The number of
up spectral reference libraries for (chemometric) classification substances in the database depends on the specific
or calibration, particular care should be taken to ensure that application. The collection of spectra in the database may be
corrections are made or measures are taken to control the represented in different ways defined by the mathematical
variability in wavenumber and response-intensity of the technique used for classification or quantitation.
instrumentation. The selectivity of the database which makes it possible to
Verification of the wavenumber scale Verify the identify positively a given material and distinguish it
wavenumber scale of the Raman shift (normally expressed in adequately from other materials in the database is to be
reciprocal centimetres) using a suitable standard which has established during the validation procedure. This selectivity
characteristic maxima at the wavenumbers under must be challenged on a regular basis to ensure ongoing
investigation, for example, an organic substance, an Ne lamp validity of the database; this is especially necessary after any
or Ar+ plasma lines from an argon-ion laser. major change in a substance (e.g. change in supplier or in the
The calibration measurement should be matched to the manufacturing process of the material) or in the set-up of the
sample type, i.e. a solid calibration sample should be used for Raman instrument (e.g. verification of the wavenumber and
solid samples and a liquid calibration sample for liquid response repeatability of the spectrometer).
samples. Choose a suitable substance (e.g. indene, This database is then valid for use only with the originating
cyclohexane or naphthalene) for which accurate wavenumber instrument, or with a similar instrument, provided the
shifts have been established (see Table 2.2.48.-1). transferred database has been demonstrated to remain valido
The indene sample can favourably be placed in an NMR Method Prepare and examine the sample in the same
tube, evacuated and sealed under inert gas, and stored cool manner as for the establishment of the database. A suitable
in the dark to avoid degradation of the sample. mathematical transformation of the Raman spectrum may be
calculated to facilitate spectrum comparison or quantitative
Table 2.2.48.-1. - Wavenumber shifts (and acceptable prediction.
toleran ces) of cyclohexane, indene and naphthalene. Comparison of the speCtra or transforms of the spectra or
cyclohexane A indene B naphthaIene A quantitative prediction of properties or amounts in the
3056.4 (± 1.5)
material in question may involve the use of a suitable
chemometric or statistical classification or calibration
2938.3 (± 1.5) technique.
2923.8 (± 1.5)
2852.9 (± 1.5)
1609.7 (± 1.0) 1576.6 (± 1.0) J. Flow Cytometry
1444.4 (± 1.0) 1552.6 (± 1.0) 1464.5 (± 1.0)
1266.4 (± 1.0) 1205.2 (± 1.0) 1382.2 (± 1.0) Flow cytometry consists of a multiparametric analysis of
1157.6 (± 1.0) 1147.2 (± 1.0) optical properties of individual parricles in a fluidic system.
1021.6 (± 1.0)
Cells or particles in suspension are individually distributed
1028.3 (± 1.0) 1018.6 (± 1.0)
into a linear array (stream), which flows through a detection
801.3 (± 1.0) 730.5 (± 1.0) 763.8 (± 1.0) device. Solid tissues have to be reduced to a single-cell
533.9 (± 1.0) 513.8 (± 1.0) suspension to be analysed.
AStandard guide {or Raman shi{t standards {or spectrometer The spectrum of parameters measurable by flow cytometry
calibration (American Society lor Testing and Materials ASTM E 1840). includes volume and morphological compléxity of cells or
B D. A. Carter, W. R. Thompson, C. E. Taylor and J. E. Pemberton, cell-like structures, cell pigments, DNA content, RNA
Applied Spectroscopy, 1995,49 (11), 1561-1576. content, proteins, cell surface markers, intracellular markers,
enzymatic activity, pH, membrane and fluidity.
Verification of the response-intensity scale The 1t is possible to collect 2 morphological parameters plus 1 or
absolute and relative intensities of the Raman bands are
more fiuorescence signals per single cel!. The multiparametric
affected by several factors including: analysis allows the definition of cell populations by their
- the state of polarisation of the irradiating light, phenotype.
- the state of polarisation of the collection optics,
V-A182 Appendix II J 2014
APPARATUS - the amount scattered at 90 0 from the direction of the light

Focusing, magnifying, and choice of Iight source are beam (si de scatter (SS)).
optimised to allow the automatic detection and measurement Forward scatter correlates with the cell volume while side
of morphological differences and staining patterns. Flow scatter is influenced by parameters such as the shape of the
cytofluorimetric analysis meets the following criteria: nucleus, the amount and type of cytoplasmic granules or the
- choice of Iight source depending on the parameters to be membrane roughness, and correlates with the morphological
analysed; complexity of the cell, so that the higher the SS intensity, the
higher the cell complexity. As a function of the
- adjustment of instrument settings depending on the cell
morphological characteristics of cells, scatter signals will
type to be analysed (for example, cell cultures, leucocytes,
always be generated during a flow analysis; they are defined
platelets, bacteria, spermatozoa, yeast) and the analysis to
as intrinsic parameters.
be performed (for example, phenotyping, cell cycle,
apoptosis, cytokines, membrane fluidity, fluorescent Fluorescence Depending on the type and number of light
protein). sources, when a cell passes through the sensing area, it will
emit fluorescent Iight. Fluorescence signals are generated
Flow cytometry is characterised by the automated
from fluorescent dyes naturally present in the cells (for
quantification of set parameters for a high number of single
example, co-enzymes, chlorophyll, seaweed pigments) anci/or
cells during ,each analysis session. For example,
from fluorescent probes taken up by the cells when stained
100 000 particles or more (practically unlimited) are analysed
for the analysis of specific characteristics (for example,
one after the other, typically in about 1 min. The detection
fluorescent antibodies, nucleic acid dyes, pH probes, calcium
limit is as low as 100 fluorescent molecules per cell.
probes, fluorescent proteins). Nowadays, there is a large
A flow cytometer apparatus has 5 main components: number and a wide range of different types of fluorescent
- a fluidic system and a flow cell; probes available. The optical filters must be adapted to the
- a light source; fluorochromes used and changed if necessary. Each
- a detection and Analogue to Digital Conversion (ADC) fluorescent probe is characterised by its excitation spectrurn
system; and its emission spectrum. They are chosen depending on
the nature of the excitation source and the detection system,
- an 'amplification system;
and according to the specific purpose of the analysis.
- a computer provided with software for analysis of the
Signal Management and Analogue to Digital
signals.
Conversion
Fluidic system and Flow Cell
Scatter and fluorescence signals emitted by cells when
The single cell is exposed to the Iight source and detected in passing across the laser beam are sorted and addressed 10
the flow cel!. The fluidic system carries the suspended cells their detectors using optical filters. The detectors are
individually from the sample tube to the laser intercept point. transducers (photomultiplier tubes (PMTs)) that convert
To achieve this, the sample stream is drawn out to a very Iight signals radiated from the cells into voltage pulses.
thin fluid thread by a sheath fluid in the flow cell
The process of counting each pulse in the appropriate
(hydrodynamic focusing). The light beam is focused in an
channel is known as Analogue 10 Digital Conversion (ADC).
elliptical shape, by 2 confocal lenses, into the flow cell
The process is finally shown as a frequency histogram.
channel through which the cells pass. The flow rate must be
constant during routine cell surface marker analysis and must Amplification Voltage pulses need to be amplified for
ensure a suitable distance between the cells to allow optimal visualisation. The amplification process accentuates
counting. the differences between cell signals, and consequently
increases the resolution among cell populations oE different
Light Sources
characteristics (for example, the differentiation of viable from
Commonly used light sources are: non-viable cells, or non-specific fluorescence from antigen-
- lamps (mercury, xenon); specific fluorescence after staining with a fluorescent
- high power water-cooled lasers (argon, krypton, dye laser); monoclonal antibody).
- low power air-cooled lasers (argon (488 nm), red helium- There are 2 methods of amplification: linear or logarithmic;
neo n (633 nm), green helium-neon, helium-cadmium the choice between the 2 types is made for every single signal
(UV)); according to the morphological characteristics of the cells and
- diode lasers (blue, green, red, violet). the staining reagents used (for example, fluorescent
monoclonal antibodies, nucleic acid dyes) .
Signal Detection
Linear amplification, which enhances the differences among
When a particle passes across the light beam, it scatters sorne
strong pulses, is used with those parameters thar' generate
of the light in all directions. Fluorescent dyes, when added to
high intensity signals, for example:
the particle, give off their own Iight (fluorescence), which is
also radiated in all directions. 2 types ofsignals may thereby - cell scatters;
be generated: - fluorescence from nucleic acid dyes for cell cycle studies.
- scatter of Iight; L ogarithmic amplification, in contrast, is for weak pulses and
- fluorescence emission. parameters or analysis conditions that may genera te both
weak and strong pulses, for example:
The instrument's Iight detectors collect sorne of this scattered
and fluorescent Iight and produce electronic signals - cell antigens;
proportional to the amount of light collected. - scatter from platelets, bacteria, yeast;
Scatter 2 parameters of light scattering are measured: - ftuorescence from nucleic acid dyes for apoptosis studies.
- the amount scattered mainly forward (forward scatter Compensation of ftuorescence signals Each ftuorescent dye
(FS)) has an absorption wavelength spectrum and a higher
2014 Appendix II K V-A183
emission wavelength spectrum. When using 2 or more Afterwards the fiuorescence plots are gated into the selected
fiuorescent pro bes simultaneously for staining cells (for region.
example, 4-antigen immunophenoryping), the fiuorochromes The analysis software allows the creation of multiple gate
emission spectra may overlap. As a consequence, each regions, using a sequentiallogic order. This feature is
fiuorescence detector will sense its own specific fiuorescent especially useful when studying rare cell populations or for
light and a variable amount of light emitted by the other sorting purposes.
fiuorescent probes. This results in signal over-evaluation and
CONTROLS
poor separation of the cell populations.
Internal control The system's optical alignment must be
The solution is in the use of an electronic matrix that allows
validated before analysis using adapted fiuorospheres and the
the selective subtraction of the interfering signals from each
optimum fiuidic stabiliry is checked. The data obtained are
fiuorescence signal after detector sensing (fiuorescence
reported and allow the periodical review of control values
compensation) .
against the mean performance value. A positive control is
Fluorescence compensation requires the use of fiuorescence highly desirable to prove that the test antibody is functional
calibrators, preferably positive cell samples stained with the and to allow the proper setting of the fiow cytometer.
fiuorochromes of interest, combined in a manner equivalent The positive control must include samples known to be
to that for the antibody used for the analysis. positive for the marker of interest.
Signal Plotting and Display External control To ensure reliabiliry in the data obtained
After amplification and compensation, the signals are plotted or to check inter-laboratory reproducibiliry, participation in a
in 2 or 3 dimensions. Histograms show the signal intensities proficiency testing study is recommended.
versus the cell counts for a given parameter. Cytograms, in
which each dot represents a cell, result from the combination
of 2 signal intensities (dual-parameter dot plots). The type
and number of plots and signal combinations are chosen on K. Peptide Identification by Nuclear
the basis of the specimens and dyes used . When analysing
acquired data, the fiow cytometry software can also generate Magnetic Resonance Spectrometry
other kinds of graphs (such as overlays, surface plots, (Ph . Eur. method 2.2.64)
tomograms, contour plots, densiry plots, overlay plots). This general chapter is to be used in conjunction with
Statistical data such as mean fiuorescent intensities (and their general chapter 2.2.33. Nuclear magnetic resonance spectrometly
shifts in time or their dependence on cell function) can also in the context of peptide identification. The approach to be
be used. followed is qualitative and consists of comparing the nuclear
Data Analysis magnetic resonance (NMR) spectrum of a test sample with
Different kinds of cell populations may be present inside the that of a reference sample acquired under identical
cell suspensions to be analysed, sorne of which are unwanted conditions.
(such as dead cells, debris or macro-aggregates), or simply This general chapter mainly applies to the use of proton
not relevant for the analysis (for example, granulocytes when NMR (lH NMR) spectrometry, to confirm the identiry of
studying lymphocytes). This depends on the cell sample type small peptide products (up to approximately 15 amino
(whole blood, bone marrow, cell cultures, biological fiuids, acids). It is also applicable when using l3C NMR
cell suspensions from solid tissues) and on the handling spectrometry with sorne modifications. The scope is
procedures (for example, staining methods, lysis, fixation, restricted to the use of one-dimensional NMR spectrometry.
cryopreservation, thawing, paraffin-embedded tissue GENERAL PRINCIPLES
preparation) . Equipment Unless otherwise specified, an apparatus with
As a consequence, not all the signals generated during a fiow a field strength giving an operating frequency for proton
cytometry analysis belong to the cells to be studied. NMR of at least 300 MHz.
2 strategies are adopted to exclude unwanted and irrelevant Spectral acquisition conditions and their optirnisation
cell signals. After introduction into the magnet, the sample is allowed to
The 1st is used during data acquisition. It is a noise come to thermal equilibrium, especially if analysis is carried
threshold, applied to 1 (or more) significant parameter(s), set out at a temperature significantly different from room
to acquire only the cells with signal intensities higher than the temperature: monitoring the lock signal is often a valuable
pre-defined discrimination value for that parameter. Due to visual guide to the progress of this process.
its characteristics of a strong signal with a low grade of The spectral width must encompass the complete spectrum
interference, forward scatter is the parameter most often used of the peptide, with an empry spectral regio? at each side.
as discriminator. Typically, a spectral width of 12 ppm or 16 ppm is
The 2nd , applied during data analysis, consists of the use of appropriate .
gating regions to restrict the analysis only to signals from The following parameters may be optimised to improve
those populations that satisfy given morphological and resolution of characteristic peaks: temperature ami/or pH
expression profile characteristics. 2 types of logical gating are primarily, buffer and peptide concentrations. Control of
commonly used. The 1s t is the morphological gateo The cell sample temperature is recommended but is not mandatory;
populations are identified using their morphological signals if not used, the effect of small temperature changes on the
(FS and SS). A region gate is drawn around the population appearance of the spectrum is validated.
of interest (for example, Iymphocytes, viable cells) then the
The number of data points collected is such as to define
fiuorescence plots are gated into the selected region. The 2nd
peaks adequately.
is the fiuorescence-based gateo The cell population of interest
is identified on the basis of the expression intensiry of an Solvent suppression is not recommended but, if used, the
antigen or a dye, then a gate region is drawn around it. intensities of peaks close to the solvent resonance may be
affected and this has to be validated when comparing spectra.
V-A184 Appendix III 2014
Chemical shift referencing For samples in aqueous

solution, sodium 2,2-dimethyl-2-silapentane-5-sulfonate
(DSS), sodium 3-(trimethylsilyl)propionate (TSP) or a
Appendix III
deuterated analogue (TSP-d 4 ) are appropriate, and the
chemical shift of the methyl signals is often set to O ppm. Chromatographic Separation Techniques
Either the reference material is added at low amounts (Ph. Eur. method 2.2.46)
(10-100 ppm has been found to be appropriate) to the Chromatographic separation techniques are multi-stage
deuterated water used to dissolve the final sample, or an separation methods in which the components of a sample are
easily recognised internal resonance that is consistently distributed between 2 phases, one of which is stationary,
present (such as acetate anion) can be used as a secondary while the other is mobile. The stationary phase may be a
reference. In this case, a validation spectrum obtained under solid or a Iiquid supported on a so lid or a gel. The stationary
the same spectral conditions is used to define the chemical phase may be packed in a colurnn, spread as a layer, or
shift of the secondary standard. distributed as a film, etc. The mobile phase may be gaseous
Samp1e size Usually a few milligrams are used. If sample or liquid or supercritical fluid. The separation may be based
sizes are variable, the effects of this variation on the on adsorption, mas s distribution (partition), ion exchange,
appearance of the spectrum are validated. etc., or may be based on differences in the physico-chemical
Sample preparation The test and reference samples must properties of the molecules such as size, mass, volume, etc.
be comparable in terms of concentration, pH and buffer This chapter contains definitions and calculations of common
composition. Typically, samples in solution are Iyophilised, parameters and generally applicable requirements for system
and the dried samples dissolved in deuterated water or a suitability. Principies of separatian, apparatus and methods
buffer in deuterated water. Ir may be worthwhile to are given in the following general methods:
lyophilise a solution in deuterated water one or more times - paper chromatography (2.2.26);
('deuterium exchange') as this reduces the intensity of strong - thin-Iayer chromatography (2.2.27);
solvent signals; volatile process impurities such as ethanol
- gas chromatography (2.2.28);
will also be lost. Use of buffer for the final sample
preparation can reduce aggregation and improve spectral - Iiquid chromatography (2.2.29);
reproducibility by reducing batch-to-batch pH variation. - size-exclusion chromatagraphy (2.2.30);
Sorne probes are intolerant to high salt concentrations, but - supercritical fluid chromatography (2.2.45).
ionic strengths up to 200 mM sodium chloride are normally
tolerated. High salt concentrations tend to increase 90° pulse DEFINITIONS
length. The system suitability and acceptance criteria in monographs have
VERIFICATION OF IDENTITY been set using parameters as defined below. Wirh some equipment,
Determination of key spectral factors Use of a certain parameters, such as the signal-to-noise ratio and resolution,
qualitative approach do es not entail stringent requirements can be calculated using software provided by the manufacturero
on spectral parameters (for example, fast pulse repetition It is the responsibility of the user to ensure that the calculation
rates can be used, as full relaxation is not required). The use methods used in the software are equivalent to the requirements of
of short pulse widths (for example, a 30° pulse) and fast the European Pharmacopoeia and to make any necessary
repetition rates will have no significantly deleterious effect on corrections if this is not the case.
spectra, and will allow faster acquisition of acceptable signal- CHROMATOGRAM
to-noise ratios. Variation in the pulse width and acquisition A graphical ar other representatian of detector response,
time within wide Iimits will not affect the ability to compare effiuent concentration or other quantity used as a measure of
spectra. The number of scans collected must give effiuent concentratian, versus time or volume. Idealised
appropriate signal-to-noise ratios for low intensity resonances chramatograms are represented as a sequence af Gaussian
and therefore a minimum signal-to-noise ratio of 50:1 is peaks on a baseline (Figure 2.2.46.-1).
recommended.
PEAK
Identification of characteristic resonances Ir is possible
The portion of a chramatogram recording the detector
to compare either the complete spectrum or a portion 'of it.
response when a single component (or 2 or more unresolved
Comparison of spectra of relevant samples will highlight
components) is eluted from the column.
regions of the spectrum that are distinctive, and comparison
can be constrained to these regions. It is important to define The peak may be defined by the peak area, or the peak
resonances from impurities, such as residual solvents, which height (h) and the peak width at half-height (Wh), ar the peak
may be essentially irrelevant to product quality and which height (h) and the peak width between the paints of
may vary in intensity between batches. inflection (wD. In Gaussian peaks (Figure 2.2.46.-1) there is
the following relationship:
Spectral comparison See the provisions of general
chapter 2.2.33.
RETENTION TIME ( T R )
Time required for elution of a component (Figure 2.2.46.-1,
baseline scale being in minutes).
RETENTION VOLUME (VR)
Volume of the mobile phase required for elution of a
component. Ir may be calculated from the retention time and
the flow rate (F) in millilitres per minute using the following
equation:
2014 Appendix III V-A185
Peak 1 Peak 2
ID
en
c:
o
a.
en
ID
c:::
Volume (V)
Time (t)
Figure 2.2.46.-1.
TOTAL MOBILE PHASE TIME (T1 0

In size-exc1usion chromatography, retention time of a
HOLD-UP TIME (TAl) component whose molecules are smaller than the smallest gel
Time required for elution of an unretained component pores (Figure 2.2.46.-2).
(Figure 2.2.46.-1, baseline scale being in minutes). In size- TOTAL MOBILE PHASE VOLUME (VT)
exc1usion chromatography, the symbol to (see below) is used. In size-exc1usion chromatography, retention volume of a
HOLD-UP VOLUME (VAl) component whose molecules are smaller than the smallest gel
Volume of the mobile phase required for elution of an pores. It may be calculated from the total mobile phase time
unretained component. It may be calculated from the hold- and the fiow rate (F) in miIlilitres per minute using the
up time and the fiow rate (F) in millilitres per minute using following equation:
the following equation:
vt := tt X F
RETENTION TIME OF AN UNRETAINED COMPOUND (To)

In size-exc1usion chromatography, the symbol Vo (see below) In size-exc1usion chromatography, retention time of a
is used. component whose molecules are larger than the largest gel
RETENTION FACTOR (K) pores (Figure 2.2.46.-2).
The retention factor (al so known as mass distribution ratio RETENTION VOLUME OF AN UNRETAINED COMPOUND (Vo)
(D m ) or capacity factor (k'» is defined as: In size-exc1usion chromatography, retention volume of a
component whose molecules are larger than the largest gel
k:= amount of component in stationary phase _ K Vs pores. It may be calculated from the retention time of an
amount of component in mobile phase - e VM unretained compound and the fiow rate (F) in millilitres per
minute using the following equation:
Kc distribution constant (also known as equilibrium
distribution coefficient); Vo := to x F
volume of the stationary phase;
volume of the mobile phase. DISTRIBUTION CONSTANT (Ko)
The retention factor of a component may be determined In size-exc1usion chromatography, the elution characteristics
from the chromatogram using the following equation: of a component in a particular column may be given by the
distribution constant (also referred to as distribution
coefficient), which is calculated using the following equation:
3le
o
c.
t,dVR2 •
tR ,IVR1
'"
~
LC and GC o
Ü
*
O
t",N", •
Size-exclusion
chromatography
107
10·",
g¡
E
105~
i3 Q)
"O
10,:2
10'
Time (t)
Volume (V)
.......1 - - - - t/V,
Figure 2.2.46.-2.
RETARDATION FACTOR (RFl

The retardation factor (also known as retention factor (R¡)),
used in planar chromatography, is the ratio of the distance
_A from the point of application to the centre of the spot and
the distance travelled by the solvent front from the point of
application (Figure 2.2.46.-3) .
.-0- 8
RF =-
b
a a
b b migration distance of the component;

a migration distance of the solvent fronr.
PLATE NUMBER (N)
_c The column performance (apparent efficiency) may be

ca1culated from data obtained under either isothermal,
isocratic or isodense conditions, depending on the technique,
as the plate number (al so referred to as number of theoretical
A. mobile phase front B. spot c. line of application plates), using the following equation, the values of tR and Wh
being expressed in the same units:
Figure 2.2.46.-3.
retention time of the peak corresponding to the An As value of 1.0 signifies symmetry. When As > 1.0, the
component; peak is tailing. When As < 1.0, the peak is fronting.
width of the peak at half-height.
The plate number varies with the component as well as with
the column, the column temperature, the mobile phase and
the retention time.
DWELL VOLUME (D)
The dwell volume (also known as gradient delay volume) is
the volume between the point at which the eluents meet and
the top of the column. It can be determined using the
following procedure.
Column replace the chromatographic column by an
appropriate capillary tubing (e.g. 1 m x 0.12 mm) .
Mobile phase:
--d
- mobile phase A: water R;
- mobile phase B: 0.1 per cent V/V solution of acetone R;
Time MobUe phase A MobUe phase B

(min) (per cent V(11) (per cent V(11)
Figure 2.2.46.-5
0·20 100"-7 O 0"-7100
20·30 O 100
RESOLUTION (R s )
The resolution between peaks of 2 components (Figure
2.2.46.-1) may be calculated using the following equation:
Flow rate set to obtain sufficient back-pressure
(e.g. 2 mUmin).
1.18 (tR2 - tRl)
Detection spectrophotometer at 265 nm. Rs = ---'-----'-------'-
Whl + Wh2
Determine the time (tO.5) in minutes when the absorbance
has increased by 50 per cent (Figure 2.2.46.-4).
tR2 > tRI
tRj, tR2 = retention times of the peaks;
D = tD X F
Whl> Wh2 = peak widths at half-height.
tO.5 - O.5tG (in minutes); In quantitative planar chromatography, using densitometry,
pre-defined gradient time (= 20 min); the migration distances are used instead of retention times
ftow rate (in millilitres per minute). and the resolution between peaks of 2 components may be
calculated using the following equation:
retardation factors of the peaks;

peak widths at half-height;
migration distance of the solvent front.
PEAK-TO-VALLEY RATIO (P/V)
The peak-to-valley ratio may be employed as a system
suitability criterion in a test for related substances when
lime baseline separation between 2 peaks is not achieved (Figure
tO.5
2.2.46.-6).
Figure 2.2.46.-4 H
p/v = --..E
H v
SYMMETRY FACTOR (As)
The symmetry factor of a peak (Figure 2.2.46.-5) is
height above the extrapolated baseline of the minor
peak;
A _ WO.05
height above the extrapolated baseline at the lowest
s - 2d point of the curve separating the minor and major
peaks.
WO.05 width of the peak at one-twentieth of the peak
height;
d distance between the perpendicular dropped from
the peak maximum and the leading edge of the
peak at one-twentieth of the peak height.
V-A188 Appendix nI 2014
h range of the noise in a chromatogram obtained after

injection or application of a blank, observed over a
distance equal 10 at least 5 times the width at half-
height of the peak in the chromatogram obtained with
the prescribed reference solution and, if possible,
situated equally around the place where this peak
would be found.
Figure 2.2.46.-6
Figure 2.2.46.-7.
RELATIVE RETENTION (R)
Relative retention is calculated as an estimate using the SYSTEM REPEATABILITY
following equation:
The repeatability of response is expressed as an estimated
percentage relative standard deviation (srC%» of a
consecutive series of measurements for not fewer than 3
injections or applications of a reference solution, and is
retention time of the peak of interest;
retention time of the reference peak (usually the peak
corresponding to the substance to be examined); 100 ¿ (Yi _ y)2
Sr (%) = --=-
y n-l
hold-up time.
The unadjusted relative retention (rG) is calculated using the
following equation: Yi individual values expressed as peak area, peak height,
or ratio of areas by the internal standardisation
method;
y mean of individual values;
n number of individual values.
Unless otherwise indicated, values for relative retention stated
in monographs correspond to unadjusted relative retention.
SYSTEM SUITABlLITY
In planar chromatography, the retardation factors RF " The various components of the equipment employed must be
qualified and be capable of achieving the performance
and RFi are used instead of tRsc and tRi'
required to conduct the test or assay.
SIGNAL-TO-NOISE RATIO (S /N)
The system suitability tests represent an integral part of the
The short-term noise influences the precision of
method and are used 10 ensure adequate performance of the
quantification. The signal-to-noise ratio is calculated using
chromatographic system. Apparent efficiency, retention factor
the following equation:
(mass distribution ratio), resolution, relative retention and
symmetry factor are the parameters that are usually employed
S/N = 2H in assessing the performance of the column. Factors that may
h affect the chromatographic behaviour include:
- the composition, ionic strength, temperature and apparent
H height of the peak (Figure 2.2.46.-7) corresponding to pH of the mobile phase;
the component concerned, in the chromatogram
- fiow rate, column dimensions, column temperature and
obtained with the prescribed reference solution,
pressure;
measured from the maximum of the peak to the
extrapolated baseline of the signal observed over a - siationary phase characteristics including type of
distance equal to at least 5 times the width at half- chromatographic support (particle-based or monolithic),
height; particle or macropore size, porosity, specific surface area;
- reversed-phase and other surface-modification of the
stationary phases, the extent of chemical modification (as
expressed by end-capping, carbon loading etc.).
The following requirements and any supplementary The chromatographic conditions described have been
requirements given in the individual monograph are to be validated during the elaboration of the monograph.
fulfilled unless otherwise prescribed: The system suitability tests are included to verify that the
- in a related substances test or assay, for a peak in the separation required for satisfactory performance of the test or
chromatogram obtained with a reference solution used for assay is achieved. Nonetheless, since the stationary phases are
quantification, the symmetry factor is 0.8 to 1.5, unless described in a general way and there is such a variety
otherwise prescribed; available commercially, with differences in chromatographic
- in an assay of an active substance where the value is behaviour, sorne adjustments of the chromatographic
100 per cent for apure substance, the maximum conditions may be necessary to achieve the prescribed system
permitted relative standard deviation (sr(%)max) for the suitability requirements. With reversed-phase liquid
defined limits is calculated for a series of injections of the chromatographic methods in particular, adjustment of the
reference solution using the following equation: various parameters will not always result in satisfactory
chromatography. In that case, it may be necessary to replace
the column with another of the same type (e.g. octadecylsilyl
silica gel), which exhibits the desired chromatographic
behaviour. The Knowledge database on the EDQM website
usually contains information on the column(s) used during
K constant (0.349), obtained from the expression: monograph elaboration.
For critical parameters the adjustments are defined clearly in
K = 0.6 X
V2.j6
t 90 %,5 in which 0.6 represents the
V2 . the monograph to ensure the system suitability.
THIN-LAYER CHROMATO GRAPHY AND PAPER
required percentage relative standard deviation
CHROMA TOGRAPHY
after 6 injections for B = 1.0;
B upper limit given in the definition of the Composition oi the mobile phase the amount of the
individual monograph minus 100 per cent; minor solvent component may be adjusted by ± 30 per cent
n number of replicate injections of the reference relative or ± 2 per cent absolute, whichever is the larger;
solution (3 ::; n ::; 6); for a minor component at 10 per cent of the mobile phase, a
Student's t at the 90 per cent probability leve! 30 per cent relative adjustment allows a range of
tgO %,n- l
(double sided) with n- 1 degrees of freedom. 7-13 per cent whereas a 2 per cent absolute adjustment
allows a range of 8-12 per cent, the relative value therefore
Unless otherwise prescribed, the maximum permitted relative being the larger; for a minor component at 5 per cent of the
standard deviation do es not exceed the appropriate value mobile phase, a 30 per cent re!ative adjustment allows a
given in Table 2.2.46.-1. This requirement does not apply to range of 3.5-6.5 per cent whereas a 2 per cent absolute
tests for related substances. adjustment allows a range of 3-7 per cent, the absolute value
being the larger in this case; no other component is altered
Table 2.2.46.-1. - Repeatability requirements by more than 10 per cent absolute.
Number of individual injections pH oi the aqueous component oi the mobile phase
± 0.2 pH, unless otherwise prescribed, or ± 1.0 pH when
3 4 5 6
non-ionisable substances are to be examined.
B (per cent) Maximum permitted relative standard deviation Concentration oi salts in the buffer component oi a
2.0 0.41 0.59 0.73 0.85 mobile phase ± 10 per cent.
0.92 1.06
Application volume 10-20 per cent of the prescribed
2.5 0.52 0.74
volume ifusing fine particle size plates (2-10 ¡..tm) .
3.0 0.62 0.89 1.10 1.27
LIQUID CHROMATOGRAPHY: ISOCRATIC ELUTION
Composition oi the mobile phase the amount of the
- in a re!ated substances test, the limit of quantification minor solvent component may be adjusted by ± 30 per cent
(corresponding to a signal-to-noise ratio of 10) is equal to relative or ± 2 per cent absolute, whichever is the larger (see
or les s than the disregard limito example above); no other component is altered by more than
Compliance with the system suitability criteria is required 10 per cent absolute.
throughout the chromatographic procedure. Depending on pH oi the aqueous component oi the mobile phase
various factors, such as the frequency of use of the procedure ± 0.2 pH, unless otherwise prescribed, or ± 1.0 pH when
and experience with the chromatographic system, the analyst non-ionisable substances are to be examined.
chooses an appropriate verification scheme to monitor this. Concentration oi salts in the buffer component oi a
mobile phase ± 10 per cent.
AD}USTMENT OF CHROMATOGRAPHIC
Flow rate ± 50 per cent; a larger adjustment is acceptable
CONDlTIONS
when changing the column dimensions (see the formula
The extent to which the various parameters of a b~low) .
chromatographic test may be adjusted to satisfy the system
Column parameters
suitability criteria without fundamentally modifying the
methods are listed below. Adjustment of conditions with Stationary phase:
gradient elutions is more critical than with isocratic elutions, - no change of the identity of the substituent of the
since it may lead to shifts in peaks to a different step of the stationary phase permitted (e.g. no replacement of C1S by
gradient, thus leading to the incorrect assignment of peaks, CS);
and to the masking of peaks or a shift such that elution - particle size: maximum reduction of 50 per cent;
occurs beyond the prescribed e!ution time. Changes other no increase permitted.
than those indicated require revalidation of the method.
Column dimensions: The isocratic step introduced for this purpose may be
- ± 70 per cent;
length: omitted if validation data for application of the method
- imemal diam eter: ± 25 per cent.
without this step is available.
pH 01 the aqueous component 01 the mobile phase no
When column dimensions are changed, the fiow rate may be
adjusted as necessary using the following equation: adjustrnent permitted.
Concentration 01 salts in the buffer component 01 a
mobile phase no adjustrnent permitted.
Flow rate adjustment is acceptable when changing the
column dimensions (see the formula below).
Column parameters
fiow rate indicated in the monograph, in millilitres per Stationary phase:
minute;
- no change of the identity of the substituent of the
adjusted fiow rate, in millilitres per minute;
stationary phase permitted (e.g. no replacement of C1S by
length of the column indicated in the monograph, in
CS);
millimetres;
length of the column used, in millimetres; - particle size: no adjustrnent permitted.
internal diameter of the column indicated in the Column dimensions:
monograph, in millimetres; -length: ± 70 per cent;
internal diameter of the column used, in millimetres. - imemal diameter: ± 25 per cent.
Ternperature ± 10 oC, where the operating temperature When column dimensions are changed, the fiow rate may be
is specified, unless otherwise prescribed. adjusted as necessary using the following equation:
Detector wavelength no adjustrnent permitted.
Injection volume may be decreased, provided detection
and repeatability of the peak(s) to be determined are
satisfactory; no increase permitted.
LIQUID CHROMATOGRAPHY: GRADIENT ELUTION
FI fiow rate indicated in the monograph, in millilitres per
Adjustrnent of chromatographic conditions for gradient minute;
systems requires greater caution than for isocratic systems. F2 adjusted fiow rate, in millilitres per minute;
Composition 01 the mobile phase/gradient elution 11 length of the column indicated in the monograph, in
minor adjustrnents of the composition of the mobile phase millimetres;
and the gradient are acceptable provided that: 12 length of the column used, in millimetres;
- the system suitability req'uirements are fulfilled; di intemal diameter of the column indicated in the
- the principal peak(s) elute(s) within ± 15 per cent ofthe monograph, in millimetres;
indicated retention time(s); d2 intemal diameter of the column used, in millimetres.
- the final composition of the mobile phase is not weaker in Teinperature ± 5 oC, where the operating temperature is
elution power than the prescribed composition. specified, unless otherwise prescribed.
Where compliance with the system suitability requirements Detector wavelength no adjustrnent permitted.
cannot be achieved, it is ofren preferable to consider the Injection volume may be decreased, provided detection
dwell volume or ro change the column. and repeatability of the peak(s) to be determined are
Dwell volume The configuration of the equipment satisfactory; no increase permitted.
employed may significantly alter the resolution, retention GASCHROMATOGRAPHY
time and relative retentions described. Should this occur, it Column parameters
may be due to excessive dwell volume. Monographs
Stationary phase:
preferably inc1ude an isocratic step before the start of the
gradient programme so thar an adaptation can be made to - particle size: maximum reduction of 50 per cent;
the gradient time points to take account of differences in no increase permitted (packed columns);
dwell volume between the system used for method - film thickness: - 50 per cent 10 + 100 per cent (capillary
development and that actually used. It is the user's columns).
responsibility to adapt the length of the isocratic step to the Column dimensions:
analytical equipment used. If the dwell volume used during - length: ± 70 per cent;
the elaboration of the monograph is given in the monograph,
the time points (t min) stated in the gradienr table may be - ± 50 per cent.
intemal diameter:
replaced by adapted time points (tc min), calculated using Flow rate ± 50 per cent.
the following equation: Ternperature ± 10 per cent.
Injection volume and split volume may be adjusted,
(D - Do) provided detection and repeatability are satisfactory.
te = t - F
SUPERCRITICAL FLUID CHROMATOGRAPHY
Composition 01 the mobile phase for packed columns,
D dwell volume, in millilitres; the amount of the minor solvent component may be adjusted
Do dwell volume used for development of the method, in by ± 30 per cent relative or ± 2 per cent absolute,
millilitres; whichever is the larger; no adjustrnent is permitted for a
F fiow rate, in millilitres per minute. capillary column system.
Detector wavelength no adjustrnent permitted.
2014 Appendix In A V-A191
Column parameters factors indicated in the monograph are applied (i.e. when the
Stationary phase: response factor is outside the range 0.8-1.2).
- particle size: maximum reduction of 50 per cent; When the related substances test prescribes the total of
no increase permitted (packed columns). impurities or there is a quantitative determination of an
impurity, it is important to choose an appropriate threshold
Column dimensions:
setting and appropriate conditions for the integration of the
-length: ± 70 per cent; peak areas. In such tests the disregard limit, i.e. the limit at or
- internal diameter. below which a peak is disregarded, is generally 0.05 per cent.
± 25 per cent (packed columns); Thus, the threshold setting of the data collection system
± 50 per cent (capillary columns). corresponds to at least half of the disregard limit. Integration
of the peak area of any impurity that is not completely
Flow rate ± 50 per cent.
separated from the principal peak is preferably performed by
Temperature ± 5 oC, where the operating temperature is valley-to-valley extrapolation (tangential skim) .
specified.
Additional points lor monographs 01 the British
Injection volume may be decreased, provided detection
Pharmacopoeia
and repeatability are satisfactory; no increase permitted.
System suitability
QUANTIFICATION Unless otherwise stated in the monograph, the maximum
Peaks due to solvents and reagents or arising from the mobile permitted relative standard deviation for six replicate
phase or the sample matrix are disregarded during injections of the prescribed reference solution does not
quantification. exceed 2.0%. This requirement is applicable only to assays.
- Detector sensitivity. The detector sensitivity is the signal
output per unit concentration or unit mas s of a substance
in the mobile phase entering the detector. The relative
detector response factor, commonly referred to as response A. Thin-Iayer Chromatography
factor, express es the sensitivity of a detector for a given (Ph. Eur. method 2.2.27)
substance relative to a standard substance. The correction Thin-Iayer chromatography is a separation technique in
factor is the reciprocal of the response factor. which a stationary phase consisting of an appropriate material
- External standard method. The concentration of the is spread in a uniform thin layer on a support (plate) of glass,
componentes) to be analysed is determined by comparing metal or plastic. Solutions of analytes are deposited on the
the responsees) (peak(s)) obtained with the test solution to plate prior to development. The separation is based on
the responsees) (peak(s)) obtained with a reference adsorption, partition, ion-exchange or on combinations of
solution. these mechanisms and is carried out by migration
- Internal standard method. Equal amounts of a component (development) of solutes (solutions of analytes) in a solvent
that will be resolved from the substance to be examined or a suitable mixture of solvents (mobile phase) through the
(the internal standard) are introduced into the test thin-Iayer (stationary phase).
solution and a reference solution. The internal standard is
chosen such that it do es not react with the substance to Apparatus
be examined, is stable and do es not contain impurities Plates The chromatography is carried out using pre-coated
with the same retention time as that of the substance to plates as described under Reagents (4.1.1).
be examined. The concentration of the substance to be Pre-treatment of the plates It may be necessary to wash
examined is determined by comparing the ratio of the the plates prior to separation. This can be done by migration
peak areas or peak heights due to the substance to be of an appropriate solvent. The plates may also be
examined and the internal standard in the test solution impregnated by procedures such as development, immersion
with the ratio of the peak areas or peak heights due to the or spraying. At the time of use, the plates may be activated,
substance to be examined and the internal standard in the if necessary, by heating in an oven at 120 oC for 20 mino
reference solution. Chromatographic tank with a flat bottom or twin trough,
- Normalisation procedure. The percentage content of a of inert, transparent material, of a size suitable for the plates
component of the substance to be examined is calculated used and provided with a tightly fitting lid. For horizontal
by determining the area of the corresponding peak as a development the tank is provided with a trough for the
percentage of the total area of all the peaks, excluding mobile phase and it additionally contains a device for
those due to solvents or reagents or arising from the directing the mobile phase to the stationary phase.
mobile phase or the sample matrix, and those at or below Micropipettes, microsyringes, calibrated disposable
the disregard limit. capillaries or other application devices suitable for the
- Calibration procedure. The relationship between the proper application of the solutions.
measured or evaluated signal (y) and the quantity Fluorescence detection device to measure direct
(concentration, mas s, etc.) of substance (x) is determined fluorescence or the inhibition of fluorescence .
and the calibration function is calculated. The analytical
Visualisation devices and reagents Suitable devices are
results are calculated from the measured signal or
used for derivatisation to transfer to the plate reagents by
evaluated signal of the analyte by means of the inverse
spraying, irnmersion or exposure to vapour and, where
function.
applicable, to facilitate heating for visualisation of separated
In tests for related substances for both the external standard components.
method, when a dilution of the test solution is used for
Documentation A device may be used to provide
comparison, and the normalisation procedure, any correction
documentation of the visualised chromatogram, for example
a photograph or a computer file.
V-Al92 Appendix In A 2014
METHOD Verification of the separating power for identification

Sample application Apply the prescribed volume of the Normally the performance given by the suitability test
solutions at a suitable distance from the lower edge and from described in Reagents (4.1.1) is sufficient. Only in special
the sides of the plate and on a line parallel to the lower edge; cases an additional performance criterion is prescribed in the
allow an interval of at least 10 mm (5 mm on high- monograph.
performance plates) between the centres of circular spots and Related substances test The secondary spot(s) in the
5 mm (2 mm on high-performance plates) between the chromatogram obtained with the test solution is (are) visually
edges of bands. Apply the solutions in sufficiently small compared to either the corresponding spot(s) in the
portions to obtain circular spots 2-5 mm in diameter chromatogram obtained with the reference solution
(1 -2 mm on high-performance plates) or bands 10-20 mm containing the impurity(ies) or the spot in the chromatogram
(5-10 mm on high-performance plates) by 1-2 mm. obtained with the reference solution prepared from a dilution
In a monograph, where both normal and high-performance of the test solution.
plates may be used, the working conditions for high- Verification ofthe separating power The requirements
performance plates are given in the brackets [ 1 after those for the verification of the separating power are prescribed in
for normal plates. the monographs concemed.
Vertical development Line the walls of the Verification ofthe detecting power The detecting power
chromatographic tank with filter paper. Pour into the is satisfactory if a spot or band is clearly visible in the
chromatographic tank a sufficient quantity of the mobile chromatogram obtained with the most dilute reference
phase for the size of the tank to give after impregnation of solution.
the filter paper a layer of appropriate depth related to the
dimension of the plate to be used. For saturation of the Quantitative measurement
chromatographic tank, replace the lid and allow to stand at The requirements for resolution and separation are
20-25 oC for 1 h. Unless otherwise indicated in the prescribed in the monographs concemed.
monograph, the chromatographic separation is performed in Substances separated by thin-Iayer chromatography and
a saturated tank. Apply the prescribed volume of solutions as responding to UV-Vis irradiation can be determined directly
described aboye. When the solvent has evaporated from the on the plate, using appropriate instrumentation. While
applied solutions, place the plate in the chromatographic moving the plate or the measuring device, examine the plate
tank, ensuring that the plate is as vertical as possible and by measuring the refiectance of the incident light. Similarly,
that the spots or bands are aboye the surface of the mobile fiuorescence may be measured using an appropriate optical
phase. Close the chromatographic tank, maintain it at system. Substances containing radionuclides can be
20-25 oC and protect from sunlight. Remove the plate when quantified in 3 ways: either directly by moving the plate
the mobile phase has moved over the prescribed distance, alongside a suitable counter or vice versa (see
measured between the points of application and the solvent Radiopharmaceutical preparations (0125)), by cutting the plates
front. Dry the plate and visualise the chromatograms as into strips and measuring the radioactivity on each individual
prescribed. strip using a suitable counter or by scraping off the stationary
For two-dimensional chromatography, dry the plates after the phase, dissolving ir in a suitable scintillation cocktail and
first development and carry out a second development in a measuring the radioactivity using a liquid scintillation
direction perpendicular to that of the first development. counter.
Horizontal development Apply the prescribed volume of Apparatus The apparatus for direct measurement on the
the solutions as described aboye. When the solvent has plate consists of:
evaporated ftom the applied solutions, introduce a sufficient - a device for exact positioning and reproducible dispensing
quantity of the mobile phase into the trough of the chamber of the amount of substances onto the plate;
using a syringe or pipette, place the plate in the chamber
- a mechanical device to move the plate or the measuring
after verifying that the latter is horizontal and connect the
device along the x-axis or the y-axis;
mobile phase direction device according to the
manufacturer's instructions . If prescribed, develop the plate - a recorder and a suitable integrator or a computer;
starting sirnultaneously at both ends. Close the chamber and - for substances responding to UV- Vis irradiation: a photometer
maintain it at 20-25 oC. Remove the plate when the mobile with a source of light, an optical device able to generate
phase has moved over the distance prescribed in the monochromatic Iight and a photo cell of adequate
monograph. Dry the plate and visualise the chromatograms sensitivity are used for the measurement of refiectance or
as prescribed. transmittance; if fiuorescence is measured, a suitable filter
For two-dimensional chromatography, dry the plates after the is required to prevent Iight used for excitation from
first development and carry out a second development in a reaching the detector while perrnitting emitted light or a
direction perpendicular to that of the first development. specific portion thereof to pass;
- for substances containing radionuclides: a suitable counter for
Visual evaluation radioactivity. The linearity range of the counting device is
Identification The principal spot in the chromatogram to be verified.
obtained with the test solution is visually compared to the Method Prepare the solution of the substance to be
corresponding spot in the chromatogram obtained with the examined (test solution) as prescribed in the monograph
reference solution by comparing the colour, the size and the and, if necessary, prepare the reference solutions of the
retardation factor (RF ) of both spots. substance to be determined using the same solvent as in the
The retardation factor (R F ) is defined as the ratio of the test solution. Apply the same volume of each solution to the
distance from the point of application to the centre of the plate and develop.
spot and the distance travelled by the solvent front from the Substances responding to UV-Vis irradiation Prepare
point of application. and apply not fewer than 3 reference solutions of the
2014 Appendix III A V-A193
substance 10 be examined, the concentrations of which span Ultraviolet Ray Lamps for Analytical Purposes
the expected value in the test solution (about 80 per cent, (Ph. Eur. text 2.1.3)
100 per cent and 120 per cent). Treat with the prescribed Mercury vapour in quartz lamps is used as the source of
reagent, if necessary, and record the reflectance, the ultraviolet light. A suitable filter may be fined 10 eliminate
transminance or fluorescence in the chromatograms obtained the visible part of the spectrum emined by the lamp. When
with the test and reference solutions. Use the measured the Pharmacopoeia prescribes in a test the use of ultraviolet
results for the calculation of the amount of substance in the light of wavelength 254 nm or 365 nm, an instrument
test solution. consisting of a mercury vapour lamp and a filter which gives
Substances containing radionuclides Prepare and apply an emission band with maximum intensity at about 254 nm
a test solution containing about 100 per cent of the expected or 365 nm is used. The lamp used should be capable of
value. Determine the radioactiviry as a function of the path revealing without doubt a standard spot of sodium salicylate
length and report the radioactivity in each resulting peak as a with a diameter of about 5 mm on a support of silica gel G R,
percentage of the total amount of radioactivity. the spot being examined while in a position normal 10 the
Criteria for assessing the suitability of the system are radiation.
described in the chapter on Chromatographic separation For this purpose apply 5 J.lL of a 0.4 giL solution of sodium
techniques (2.2.46). The extent 10 which adjustments of salicylate R in alcohol R 1 for lamps of maximum output at
parameters of the chromatographic system can be made to 254 nm and 5 J.lL of a 2 gIL solution in alcohol R 1 for lamps
satisfy the criteria of system suitability are also given in this of maximum output at 365 nm. The distance between the
chapter. lamp and the chromatographic plate under examination used
Additional points for monographs of the British in a pharmacopoeial test should never exceed the distance
Pharmacopoeia used to carry out the aboye test.
When the method prescribed in a monograph carries the Identification of Phenothiazines by Thin-Layer
instructions 'protected from light' or 'in subdued light' it is Chromatography
intended that the entire procedure is carried out under these
conditions . (Ph . Eur. method 2.3.3)
Examine by thin-Iayer chromatography (2.2.27) using
Unless otherwise indicated in the monograph, the mobile
kieselguhr G R as the coating substance. Impregnate the plate
phase should be allowed 10 ascend 15 cm aboye the line of
by placing it in a closed tank containing the necessary
application.
quantity of the impregnation mixture composed of a solution
The phrase ultraviolet light (254 nm) indica tes that the plate containing 10 per cent VIV of phenoxyethanol R and 50 gIL of
should be examined under an ultraviolet lamp having a macrogol 300 R in acetone R so that the plate dips about
maximum output at 254 nm (see below); other wavelength 5 mm beneath the surface of the liquid. When the
maxima may be specified. impregnation mixture has risen at least 17 cm from the lower
The term secondary spot means any spot other than the edge of the plate, remove the plateand use irnmediately for
principal spot. Similarly, a secondary band is any band other chromatography. Carry out the chromatography in the same
than the principal bando direc,tion as the impregnation.
Where a spraying technique is prescribed it is essential that Test solution Dissolve 20 mg of the substance to be
the reagent is evenly applied as a fine spray. The following examined in chloroform R and dilute to 10 mL with the same
method of visualisation is used when directed in the solvent.
monograph. Reference solution Dissolve 20 mg of the corresponding
METHOD 1 chemical reference substance (CRS) in chloroform R and
Spray the dried plate with ethanolic sulfuric acid (20%), heat dilute to 10 mL with the same solvent.
at 105° for 30 minutes and irnmediately expose 10 nitrous Apply separately to the plate 2 J.lL of each solution and
fumes in a closed glass tank for 15 minutes (the nitrous develop in the dark over a path of 15 cm using a mixture of
fumes may be generated by adding 7M sulfuric acid dropwise 50 mL of light petroleum R and 1 mL of diethylamine R
to a solution containing 10% w/v of sodium nitrite and saturated with phenoxyethanol R (i.e. add about 3 mL 10
3% w/v of potassium iodide). Place the plate in a current of 4 mL of phenoxyethanol R to the aboye mixture of solvents to
warm air for 15 minutes and spray with a 0.5% w/v solution give a persistent cloudiness on shaking, decant, and use the
of N-(1-naphthyl)ethylenediamine dihydrochloride in ethanol supematant liquid, even if it is cloudy). After development
(96%). If necessary, allow to dry and repeat the spraying. place the plate under ultraviolet light at 365 nm and examine
MATERIALS after a few minutes. The spot in the chromatogram obtained
The coating substances and precoated plates are described in with the test solution is similar in position, fluorescence and
Appendix 1 A: General Reagents. Prepare suspensions of the size 10 the spot in the chromatogram obtained with the
coating substances as recommended by the manufacturer reference solution. Spray with a 10 per cent V/V solution of
unless otherwise prescribed. Commercial pre-coated plates sulfuric acid R in alcohol R. The spot in the chromatogram
may be used for pharmacopoeial tests where a coating obtained with the test solution is of the same colour as that
substances is prescribed provided that they comply with the in the chromatogram obtained with the reference solution
test for chromatographic separation described for the and has similar stability over a period of at least 20 mino
corresponding coating substance and with any additional test
for verification of separating power required in the Related Substances in Phenothiazines
monograph test. (No Ph. Eur. method)
Carry out the method for thin-layer chromatography protected
from light using silica gel GF254 as the coating substance and
1 The alcohol R used mus! be free from fiu orescence.

V-A194 Appendix III B 2014
the mobile phase prescribed in the monograph, but allowing E. A mixture of equal volumes of cyclohexane and petroleum
the solvent front to ascend 12 cm aboye the line of spirit (boiling range, 40° to 60° or 50° to 70°).
application. Unless otherwise specified, apply separately to F. A mixture of 40 volumes of glacial acetic acid and
the plate 10 I1L of each of two solutions of the substance 60 volumes of water.
being examined prepared irnmediately before use in a
G. A mixture of 20 volumes of 1,4-dioxan and 80 volumes
mixture of 95 volumes of methanol and 5 volumes of
of hexane.
diethylamine containing (1) 2.0% w/v and (2) 0.010% w/v.
After removal of the plate, allow it to dry in air and examine H . A mixture of 29 volumes of toluene, 56 volumes of
under ultraviolet light (254 nm). Disregard any spot remaining chloroform and 115 volumes of cyclohexane.
on the line of application. Unless otherwise specified any
secondary spot in the chromatogram obtained with solution (1)
is not more intense than the spot in the chromatogram
obtained with solution (2) (0.5%).
B. Gas Chromatography
Mobile phases
Gas chroma1Ography (GC) is a chromatographic separation
A. A mixture of 10 volumes of acetone, 10 volumes of technique based on the difference in the disrribution of
diethylamine and 80 volumes of cyclohexane. species between two non-miscible phases in which the mobile
B. A mixture of 5 volumes of diethylamine, 10 volumes of phase is a carrier gas moving through or passing the
acetone and 85 volumes of hexane. stationary phase contained in a column. It is applicable to
C . A mixture of 18 volumes of 1M ammonia and substances or their derivatives which are volatilised under the
90 volumes of butan-I-ol. temperatures employed.
GC is based on mechanisms of adsorption, mass distribution
Identification of steroids or size exelusion.
(No Ph. Eur. method)
Carry out the method for thin-layer chromatography using Apparatus
kieselguhr G as the coating substance. Impregnate the dry The apparatus consists of an injector, a chromatographic
plate by placing it in a tank containing a shallow layer of the column contained in an oven, a detector and a data
specified impregnating solvent, allowing the solvent to ascend acquisition system (or an integrator or a chart recorder) .
to the top, removing the plilte from the tank and allowing the The carrier gas flows through the column at a conrrolled rate
solvent to evapora te; use within 2 hours, with the fiow of the or pressure and then through the detector.
mobile phase in the direction in which impregnation was The chromatography is carried out either at a constant
carried out. Unless otherwise specified, apply separately to temperature or according 10 a given temperature programme.
the plate 2 I1L of each of the following three solutions in a
mixture of 9 volumes of chloroform and 1 volume of methanol.
Injectors
Solution (1) contains 0.25% w/v of the substance being Direct injections of solutions are the usual mode of injection,
examined. Solution (2) contains 0.25% w/v of the unless otherwise prescribed in the monograph. Injection may
corresponding British Pharmacopoeia Chemical Reference be carried out either directly at the head of the column using
Substance or European Pharmacop6eia Chemical Reference a syringe or an injection valve, or into a vaporisationchamber
Substance. Solution (3) is a mixture of equal volumes of which may be equipped with a srream splitter.
solutions (1) and (2). Use the specified mobile phase. After Injections of vapour phase may be effected by static or dynamic
removal of the plate, allow the solvent to evaporate, heat at head-space injection systems.
120 0 for 15 minutes and spray the hot plate with ethanolic Dynamic head-space (purge and trap) injection systems
sulfuric acid (20%). Heat at 120 0 for a further 10 minutes, inelude a sparging device by which volatile substances in
allow 10 cool and examine in daylight and under ultraviolet solution are swept into an absorbent column maintained at a
light (365 nm) . The principal spot in the chromatogram low temperature. Retained substances are then desorbed into
obtained with solution (1) is similar in position, colour in the mobile phase by rapid heating of the absorbent column.
daylight, fiuorescence in ulrraviolet light (365 nm) and size to
Static head-space injection systems inelude a thermostatically
that in the chroma1Ogram obtained with solution (2).
conrrolled sample heating chamber in which elosed vials
The principal spot in the chromatogram obtained with containing solid or liquid samples are placed for a fixed
solution (3) appears as a single, compact spot.
period of time to allow the volatile components of the sample
Impregnating solvents to reach equilibrium between the non-gaseous phase and the
1. A mixture of 1 volume of formamide and 9 volumes of vapour phase. After equilibrium has been established, a
acetone. predetermined amount of the head-space of the vial is
n. A mixture of 1 volume of propane-I,2-diol and 9 volumes flushed into the gas chromatograph.
of acetone. Stationary phases
III. A mixture of 1 volume of liquid paraffin and 9 volumes Stationary phases are contained in columns which may be:
of petroleum spirit (boiling range, 40° to 6(f' or 50° to 70°). - a capillary column of fused-silica whose wall is coated
Mobile phases with the stationary phase,
A. Chloroform. - a column packed with inert particles impregnated with the
stationary phase,
B. A mixture of 25 volumes of chloroform and 75 volumes
of toluene. - a column packed with solid stationary phase.
C. Toluene. Capillary columns are 0.1 mm to 0.53 mm in internal
D. A mixture of 20 volumes of toluene and 80 volumes of diameter (0) and 5 m to 60 m in length. The liquid or
cyclohexane. stationary phase, which may be chemically bonded to the
inner surface, is a film 0.1 11m to 5.0 11m thick.
2014 Appendix III B V-A195
Packed columns, made of glass or metal, are usually 1 m to 3 The sample to be analysed is introduced into a container
m in length with an internal diameter (0) of 2 mm to 4 mm. fitted with a suitable stopper and a valve-system which
Stationary phases usually consist of porous polymers or solid permits the passage of the carrier gas. The container is
supports impregnated with liquid phase. placed in a thermostatically controlled chamber at a
Supports for analysis of polar compounds on columns packed temperature set according to the substance to be examined.
with low-capacity, low-polarity stationary phase must be inert The sample is held at this temperature long enough to allow
to avoid peak tailing. The reactivity of support materials can equilibrium to be established between the solid or liquid
be reduced by silanising prior to coating with liquid phase. phase and the vapour phase.
Acid-washed, fiux-calcinated diatomaceous earth is often The carrier gas is introduced into the container and, after the
used. Materials are available in various particle sizes, the prescribed time, a suitable valve is opened so that the gas
most commonly used particles are in the ranges of 150 !lm to expands towards the chromatographic column taking the
180 !lm and 125 !lm to 150 ¡.¡rn. volatilised compounds with it.
Mobile Phases Instead of using a chromatograph specifically equipped for
Retention time and peak efficiency depend on the carrier gas the introduction of samples, it is also possible to use airtight
fiow rate; retention time is directly proportional to column syringes and a conventional chromatograph. Equilibration is
length and resolution is proportional to the square root of the then carried out in a separate chamber and the vapour phase
column length. For packed columns, the carrier gas fiow rate is carried onto the column, taking the precautions necessary
is usually expressed in millilitres per minute at atrnospheric to avoid any changes in the equilibrium.
pressure and room temperature. Flow rate is measured at the
detector outlet, either with a calibrated mechanical device or Method
with a bubble tube, while the columnis at operating Using the reference preparations, determine suitable
temperature . The linear velocity of the carrier gas through a instrument settings to produce an adequate response.
packed column is inversely proportional to the square root of Direct calibration
the internal diameter of the column for a given fiow volume.
Separately introduce into identical containers the preparation
Flow rates of 60 mUmin in a 4 mm internal diameter
to be examined and each of the reference preparations, as
column and 15 mUmin in a 2 mm internal diameter
prescribed in the monograph, avoiding contact between the
column, give identicallinear velocities and thus similar
sampling device and the samples.
retention times.
Close the containers hermetically and place in the
Helium or nitrogen are usually employed as the carrier gas
thermostatically controlled chamber set to the temperature
for packed columns, whereas commonly used carrier gases
and pressure prescribed in the monograph; after
for capillary columns are nitro gen, helium and hydrogen.
equilibration, carry out the chromatography under the
Detectors prescribed conditions.
Flame-ionisation detectors are usually employed but Standard additions
additional detectors which may be used inelude: electron-
Add to a set of identical suitable containers equal volumes of
capture, nitrogen-phosphorus, mass spectrometric, thermal
the preparation to be examined. Add to all but one of the
conductivity, Fourier transform infrared spectrophotometric,
containers, suitable quantities of a reference preparation
and others, depending on the purpose of the analysis.
containing a known concentration of the substance to be
Method determined so as to produce a series of preparations
containing steadily increasing concentrations of the
Equilibrate the column, the injector and the detector at the substance.
temperatures and the gas fiow rates specified in the
Close the containers hermetically and place in the
monograph until a stable baseline is achieved. Prepare the
thermostatically controlled chamber set to the temperature
test solution(s) and the reference solution(s) as prescribed.
and pressure prescribed in the monograph; after
The solutions must be free from solid partieles.
equilibration, carry out the chromatography under the
Criteria for assessing the suitability of the system are prescribed conditions.
described in the chapter on Chromatographic separation
Calculate the linear equation of the graph using a least-
techniques (2.2.46) . The extent to which adjustrnents of
squares fit, and derive from it the concentration of the
parameters of the chromatographic system can be made to
substance to be determined in the preparation to be
satisfy the criteria of system suitability are also given in this
chapter. examined.
Alternatively, plot on a graph the mean of readings against
Sta tic head-space gas chromatography the added quantity of the substance to be determined.
Static head-space gas chromatography is a technique Extrapolate the line joining the points on the graph until it
particularly suitable for separating and determining volatile meets the concentration axis. The distance between this
compounds present in solid or liquid samples. The method is point and the intersection of the axes represents the
based on the analysis of the vapour phase in equilibrium with concentration of the substance to be determined in the
the solid or liquid phase. preparation to be examined.
Apparatus Successive withdrawals (multiple head-space

extraction)
The apparatus consists of a gas chromatograph provided with
a device for introducing the sample that may be connected to If prescribed, the successive withdrawal method is fully
a module that automatically controls the pressure and the described in the monograph.
temperature. If necessary, a device for eliminating solvents
can be added.
Additional points for monographs of the British used. The sample is introduced into a column, which is filled
Pharmacopoeia with a gel or a porous particle packing material, and is
APPARATUS carried by the mobile phase through the column. The size
The design of a particular chromatograph may require separation takes place by repeated exchange of the solute
modification of the conditions detailed in the monograph. molecules between the solvent of the mobile phase and the
In such a case, the analyst should be satisfied that the same solvent in the stagnant liquid phase (stationary phase)
modified conditions produce comparable results. If necessary, within the pores of the packing material. The pore-size range
adjust the flow rate of the carrier gas to improve the quality of the packing material determines the molecular-size range
of the chromatogram or to modifY the retention times of the within which separation can occur.
peaks of interest. Molecules small enough to penetrate all the pore spaces elute
METHOD at the total permeation volume (Vt ). On the other hand,
Unless otherwise stated in the monograph, use nitrogen as molecules apparently larger than the maxirnum pore size of
the carrier gas and a flame ionisation detector. Occasionally the packing material migrate along the column only through
reference is made to on-column injection, in which case the the spaces between the particles of the packing ·material
sample is injected directly on to the packing material without without being retained and elute at the exclusion volume (Vo
the use of an inlet heater. When non-volatile material is to be void volume) . Separation according to molecular size occurs
injected on to the column, a suitable interchangeable pre- between the exclusion volume and the total permeation
column may be used. volume, with useful separation usually occurring in the first
two thirds of this range.
REAGENTS
Apparatus The apparatus consists essentially of a
Solvents and reagents used in the preparation of solutions for
chromatographic column of varying length and intemal
examination should be of a quality suitable for use in gas
di ame ter (0), if necessary temperature-controlled, packed
chromatography. A wide range of chemical substances is
with a separation material that is capable of fractionation in
used as stationary phases, including polyethylene glycols,
the appropriate range of molecular sizes and through which
high-molecular weight esters and amides, hydrocarbons,
the eluent is passed at a constant rateo One end of the
silicone gums and fluids (polysiloxanes often substituted with
column is usually fitted with a suitable device for applying
methyl, phenyl, nitrilo, vinyl or fluoroalkyl groups or
the sample such as a flow adapter, a syringe through a
mixtures of these) and microporous cross-linked polyaromatic
septum or an injection valve and may also be connected to a
beads. A suitable stationary phase, its concentration and the
suitable pump for controlling the flow of the eluent.
nature and grade of a suitable solid support are stated in the
Alternatively the sample may be applied directly to the
monograph. The column should be conditioned in
drained bed surface or, where the sample is denser than the
accordance with the manufacturer's instructions. In most
eluent, it may be layered beneath the eluent. The outlet of
cases reference is made to a particular commercial brand that
the column is usually connected to a suitable detector fitted
has been found to be suitable for the purpose, but such
with an automatic recorder which enables the monitoring of
statements do not imply that a different but equivalent
the relative concentrations of separated components of the
commercial brand may not be used.
sample. Detectors are usually based on photometric,
INTERNALSTANDARDS refractometric or luminescent properties. An automatic
Reagents used as internal standard s should not contain any fraction collector may be attached, if necessary.
impurity that would produce a peak likely to interfere in the The packing material may be a soft support such as a swoIlen
determination described in the monograph. gel or a rigid support composed of a material such as glass,
INJECTION VOLUME silica or a solvent-compatible, cross-linked organic polymer.
Where no injection volume is specified in the monograph, Rigid supports usually require pressurised systems giving
the analyst should select an appropriate volume for their fas ter separations. The mobile phase is chosen according to
specific application. The volume chosen is dependent on the sample type, separation medium and method of detection.
response of the analyte, the detector used, the efficiency of Before carrying out the separation, the packing material is
the column and the overall performance of the treated, and the column is packed, as described in the
chromatographic system. Where a volume is not indicated, monograph, or according to the manufacturer's instructions.
1 ¡J.L is usually appropriate; however this should be checked Criteria for assessing the suitability of the system are
for suitability under the local operating conditions. described in the chapter on Chromatographic separation
SECONDARY PEAK S techniques (2.2.46). The extent to which adjustments of
Reference may be made to a secondary peak. A secondary parameters of the chromatographic system can be made to
peak is a peak in the chromatogram other than the principal satisfY the criteria of system suitability are also given in this
peak and any peaks due to internal standard, solvent and chapter.
derivatising agents. Peaks identified as being due to the Determination of relative component composition of
counter-ion and/or other excipients including preservatives in mixtures
the material being examined may also be excluded. Carry out the separation as stated in the monograph.
If possible, monitor the elution of the components
continuously and measure the corresponding peak areas.
C. Size-exclusion Chromatography If the sample is monitored by a physico-chemical propetty to
which all the components of interest exhibit equivalent
responses (for example if they have the same specific
Size-exclusion chromatography is a chromatographic
absorbance), calculate the relative amount of each
technique which separates molecules in solution according to
component by dividing the respective peak area by the sum
their size. With organic mobile phases, the technique is
of the peak areas of aIl the components of interest. If the
known as gel-permeation chromatography and with aqueous
responses to the propetty used for detection of the
mobile phases, the term gel-filtration chromatography has been
2014 Appendix III e V-A197
components of interest are not equivalent, calculate the volume Va and from the peak corresponding to dextrose,
content by means of calibration curves obtained with the calculate the total volume v,.
calibration standards prescribed in the monograph. Inject the chosen volume of each of the calibration solutions.
DETERMINATION OF MOLECULAR MASSES Draw carefully the baseline of each of the chromatograms.
Size-exclusion chromatography may be used to determine Divide each chromatogram into p (at least 60) equal vertical
molecular masses by comparison with appropriate calibration sections (corresponding to equal elution volumes). In each
standards specified in the monograph. The retention volumes section i, corresponding to an elution volume Vi measure the
of the calibration standards may be plotted against the height (y¡) of the chromatogram line aboye the baseline and
logarithm of their molecular masses. The plot usually calculate the coefficient of distribution Ki using the
approximates a straight line within the exclusion and total expression:
permeation limits for the separation medium used . From the
calibration curve, molecular masses may be estimated. (Vi - Va)
The molecular-mass calibration is val id only for the particular (Vi - Va) (1)
macromolecular solute/solvent system used under the
specified experimental conditions. Va void volume of the column, determined using the
DETERMINATION OF MOLECULAR SIZE DISTRIBUTION OF peak corresponding to dextran Va CRS in the
POLYMERS chromatogram obtained with the marker solution,
Size-exclusion chromatography may be used to determine the V, total volume of the column, determined using the
distribution of the molecular size of polymers. However, peak corresponding to glucose in the chromatogram
sample comparison may be valid only for results obtained obtained with the marker solution,
under the same experimental conditions. The reference Vi = elution volume of section i in the chromatogram
substances used for the calibration and the methods for obtained with each of the calibration solutions.
determination of the distribution of molecular sizes of Carry out the calibration using either of the following
polymers are specified in the monograph. methods.
Calibration by plotting of the curve For each of the
Molecular Mass Distribution in Dextrans
dextrans for calibration calculate the coefficient of
(Ph. Eur. method 2.2.39) distribution Kmax corresponding to the maximum height of
Examine by size-exclusion chromatography (2.2.30). the chromatographic line, using expression (1). Plot on
Test solution Dissolve 0.200 g of the substance to be semilogarithmic paper the values of Km ax (on the x-axis)
examined in the mobile phase and dilute to 10 mL with the against the declared molecular mass at the maximum height
mobile phase . of the chromatographic line (MmaJ of each of the dextrans
Marker solution Dissolve 5 mg of glucose R and 2 mg of for calibration and glucose. Draw a first calibration curve
dextran Va CRS in 1 mL of the mobile phase. through the points obtained, extrapolating it from the point
Kmax obtained with dextran 250 for calibration CRS to the
Calibration solutions Dissolve separately in 1 mL of the
lowest K value obtained for this CRS (Figure 2.2.39.-1).
mobile phase 15 mg of dextran 4 for calibration CRS, 15 mg
Using this first calibration curve, transform, for each
of dextran 10 for calibration CRS, 20 mg of dextran 40 for
chromatogram, all Ki values into the corresponding
calibration CRS, 20 mg of dextran 70 for calibration CRS and
molecular mass Mi' thus obtaining the molecular mass
20 mg of dextran 250 for calibration CRS.
distribution. Calculate for each dextran for calibration the
System suitability solution Dissolve either 20 mg of average molecular mass Mw using equation (3) below. If the
dextran 40 for performance test CRS (for dextran 40) or 20 mg calculated values for Mw do not differ by more than
of dextran 60/70 for performance test CRS (for dextran 60 and 5 per cent from those declared for each of the dextrans for
dextran 70) in 1 mL of the mobile phase. calibration and the mean difference is within ± 3 per cent,
The chromatographic procedure may be carried out using: the calibration curve is approved. If not, move the calibration
- a column 0.3 m long and 10 mm in internal diameter, curve along the y-axis and repeat the procedure aboye until
packed with cross-linked agarose for chromatography R or a the calculated and the declared values for Mw do not differ
series of columns, 0.3 m long and 10 mm in internal by more than 5 per cent.
diameter, packed with polyether hydroxylated gel for Calibration by calculation of the curve Calculate from
chromatography R, equations (2) and (3) below, using a suitable method 1,
- as the mobile phase, at a fiow rate of 0.5-1 mUmin, kept values for b¡, bl> b3 , b4 and bs that give values of M w within
constant to ± 1 per cent per hOUf, a solution containing 5 per cent of the declared values of each of the dextrans for
7 g of anhydrous sodium sulfate R and 1 g of chlorobutanol R I calibration and 180 ± 2 for glucose:
in 1 litre of water R,
- as detector a differential refractometer, (2)
- a 100 ~lL to 200 ¡..LL loop injector,
maintaining the system at a constant temperature p
(± 0.1 oC). ¿ (YiM;)
M - ;:...i=--,I,-::-_ _
W - P
Calibration of the Chromatographic System (3)
¿ Yi
Carry out replicate injections of the chosen volume of the i= 1
marker solution. The chromatogram shows 2 peaks, the first
of which corresponds to dextran Va CRS and the second of 1 An iterative merhod such as rhe Gauss-NewlOn merhod modified by
which corresponds to dextrose R. From the elution volume of Harrley is suitable (see O. Hartley, Tecnomem'cs, 3 (1961) and G. Nilsson
the peak corresponding to dextran Vo, calculate the void and K. N!lsson, J. Chromar. 101, 137 (1974)). A curve-fitting programme
jor microcomputers, capable oj non-linear regression, may be used.
n
P number of sections dividing the chromatograms, ¿ (yiMi )
Yi height of the chromatographic line aboye the baseline Mw = -'.-=i....;l:c,n=--__
in section i, ¿Yi
molecular mass in section i. i =l (4)
in which n is defined by the expressions:

M
,,
,,
,,
0 6 (5)
\
\
\
n+l
~Yi >0.1
(P)
~Yi
\
\ (6)
r,
\
\
Dextran 250
105
p number of sections dividing the chromatograms,
1.. Dextran 70 Yi height of the chromatographic line aboye the baseline
"- in section i,
Dextran40 Mi molecular mass in section i.
'\. The test is not valid unless Mw of the 10 per cent high
104 1\ Dextran 10 =1=
fraction dextran is:
- 110000 to 130000 (dextran 40 jor perfonnance test CRS),
- 190 000 10 230 000 (dextran 60/70 jor perfonnance
test CRS) .
I\. Dextran 4
Average molecular mass ofthe 10 per cent low-fraction
\ dextran. Calculate Mw for the 10 per cent low-fraction
\ dextran eluted in and after section m using the expression:
103
P
¿ (YiMi)
250
70 4
1\ Glucose/dextrose
M
W
- .:. i=_m,-,-::_ __
- P
¿
i= m
Yi
(7)
4 10 K
in which m is defined by the expressions:
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Figure 2.2.39.-1. - Example of a calibration curve.

The dofted line corresponds to the part of the curve tha! is
extrapolated. Horizontallines at the boftom ofthe figure (8)
represent the width and the position of the chromatographic
fine obtained with each of the dextrans for calibration.
The dotted tine corresponds to the part oj the curve that is

extrapolated. Horizontal lines at the bottom oj the figure represent
the width and the position oj the chromatographic fin e obtained
(9)
with each oj the dextrans jor calibration.
p number of sections dividing the chromatograms,
System Suitability Y¡ height of the chromatographic line aboye the baseline
Inject the chosen volume of the appropriate system suitability in section i,
solution. Mi molecular mass in section i.
Average molecular mass of dextran for performance The test is not valid unless M w of the 10 per cent low-
test CRS. Calculate the average molecular mass Mw as fraction dextran is:
indicated under Calibration of the chromatographic system, - 6000 10 8500 (dextran 40 jor peljormance test CRS) ,
using either the plotted calibration curve or the values
- 7000 10 11 000 (dextran 60/70 jor perfonnance test CRS).
obtained aboye for b¡, b2 , b3 , b4 and bs . The test is not valid
unless M w is: Molecular mas s distribution of the dextran to be
- 41 000 10 47 000 (dextran 40 jor performance test CRS), analysed
- 67 000 10 75 000 (dextran 60/70 jor perfonnance test CRS). Inject the chosen volume of the test solution and calculate
Average molecular mass ofthe 10 per cent high- Mw of the total molecular mass distribution, M w of the
fraction dextran Calculate Mw for the 10 per cent high- 10 per cent high-fraction dextran and M w of the 10 per cent
fractio n dextran eluted through section n using the equation: low-fraction dextran as indicated under System suitability.
2014 Appendix III D V-A199
cyc10dextrins etc., for the separation of enantiomers

D. Liquid Chromatography (chiral chromatography).
(Ph. Eur. mechad 2.2.29) Most separations are based upon partition mechanisms
Liquid chromatography (LC) is a method of utilising chemically modified silica as the stationary phase
chromatographic separation based on the difference in the and polar solvents as the mobile phase. The surface of the
distribution of species between two non-miscible phases, in support, e.g. the silanol groups of silica, is reacted with
which the mobile phase is a liquid which percolates through various silane reagents to produce covalently bound silyl
a stationary phase contained in a column. derivatives covering a varying number of active sites on the
LC is mainly based on mechanisms of adsorption, mass surface of the suppon. The nature of the bonded phase is an
distribution, ion exchange, size exc1usion or stereochemical important parameter for determining the separation
interaction. properties of the chromatographic system.
Commonly used bonded phases are shown below:
Apparatus
octyl Si-[CH 2 h-CH 3
The apparatus consists of a pumping system, an injector, a
octadecyl Si-[CH 2117 -CH 3
chromatographic column (a column temperature controller
phenyl Si-[CH 21n-C 6 H s
may be used), a detector and a data acquisition system (or an
cyanopropyl Si-[CH 2 h -CN
integrator or a chan recorder) . The mobile phase is supplied
aminopropyl Si-[CH 2h-NH 2
from one or several reservoirs and f10ws through the column,
diol Si-[CH2h-O-CH(OH)-CHz-OH
usually at a constant rate, and then through the detector.
Unless otherwise stated by the manufacturer, silica based
Pumping systems
reversed-phase columns are considered to be stable in mobile
LC pumping systems are required to deliver the mobile phases having an apparent pH in the range 2.0 to 8.0.
phase at a constant f10w rateo Pressure f1uctuations are to be Columns containing porous graphite or partic1es of polymeric
minimised, e.g. by passing the pressurised solvent through a materials such as styrene-divinylbenzene copolymer are stable
pulse-dampening device. Tubing and connections are capable over a wider pH range.
of withstanding the pressures developed by the pumping
Analysis using normal-phase chromatography with
system. LC pumps may be fitted with a facility for
unmodified silica, porous graphite or polar chemically
"bleeding" the system of entrapped air bubbles. '
modified silica, e.g. cyanopropyl or diol, as the stationary
Microprocessor controlled systems are capable of accurately phase with a non-polar mobile phase is applicable in certain
delivering a mobile phase of either constant (isocratic elution) cases.
or varying composition (gradient elution), according to a
For analytical separations, the partic1e size of the most
defined programme. In the case of gradient elution, pumping
commonly used stationary phases varies between 3 )lm and
systems which deliver solvent(s) from several reservoirs are
1O ~lm. The partic1es may be spherical or irregular, of varying
available and solvent mixing can be achieved on either the
porosity and specific surface area. These parameters
low or high-pressure side of the pump (s) .
contribute to the chromatographic behaviour of a particular
lnjectors stationary phase. In the case of reversed phases, the nature of
The sample solution is introduced into the f10wing mobile the stationary phase, the extent of bonding, e.g. expressed as
phase at or near the head of the column using an injection the carbon loading, and whether the stationary phase is end-
system which can operate at high pressure. Fixed-Ioop and capped (i.e. residual silanol groups are silylated) are
variable volume devices operated manually or by an auto- additional determining factors. Tailing of peaks, particularly
sampler are used. Manual partial filling of loops may lead to of basic substances, can occur when residual silanol groups
poorer injection volume precision. are presento
Stationary phases Columns, made of stainless steel unless otherwise prescribed
in the monograph, of varying length and internal diameter
There are many rypes of stationary phases employed in LC,
(0) are used for analytical chromatography. Columns with
inc1uding:
internal diameters of less than 2 mm are often referred to as
- silica, alumina or porous graphite, used in normal-phase microbore columns. The temperature of the mobile phase
chromatography, where the separation is based on and the column must be kept constant during an analysis.
differences in adsorption and/or mass distribution, Most separations are performed at room temperature, but
- resins or polymers with acid or basic groups, used in ion- columns may be heated to give higher efficiency. It is
exchange chromatography, where separation is based on recommended that columns not be heated aboye 60 oC
competition between the ions to be separated and those in because of the potential for stationary phase degradation or
the mobile phase, changes occurring to the composition of the mobile phase.
- porous silica or polymers, used in size-exc1usion Mobile phases
chromatography, where separation is based on differences
For normal-phase chromatography, less polar solvents are
between the volumes of the molecules, corresponding to
employed. The presence of water in the mobile phase is to be
steric exc1usion,
strictly controlled to obtain reproducible results. In reversed-
- a variety of chemically modified supports prepared from phase LC, aqueous mobile phases, with or without organic
polymers, silica or porous graphite, used in reversed-phase modifiers, are employed.
LC, where the separation is based principally on partition
Components of the mobile phase are usually filtered to
of the molecules between the mobile phase and the
remove partic1es greater than 0.45 )lm. Multicomponent
stationary phase,
mobile phases are prepared by measuring the required
- special chemically modified stationary phases, volumes (unless masses are specified) of the individual
e.g. cellulose or amylose derivatives, proteins or peptides, components, followed by mixing. Alternatively, the solvents
may be delivered by individual pumps controlled by
V -A200 Appendix III E 2014
proportioning val ves by which mixing is performed according The separation should be carried out at a constant ambient
to the desired proportion. Solvents are normally degassed temperature unless otherwise specified in the monograph.
before pumping by sparging with helium, sonication or using When using mobile phases of high pH with a silica-based
on-line membrane/vacuum modules to avoid the creation of column, it is advisable to use a pre-column before the
gas bubbles in the detector cel!. analytical column.
Solvents for the preparation of the mobile phase are normally Unless otherwise specified in the monograph the detector
free of stabilisers and are transparent at the wavelength of consists of a photometric detector fitted with a low-volume
detection, if an ultraviolet detector is employed. Solvents and ftow cell (about 10 flL is suitable); the wavelength setting is
other components employed are to be of appropriate quality. specified in the monograph.
Adjustment of the pH, if necessary, is effected using only the The design of a particular chromatograph may require
aqueous component of the mobile phase and not the modification of the conditions detailed in the monograph.
mixture. If buffer solutions are used, adequate rinsing of the In such a case the analyst should be satisfied that the
system is carried out with a mixture of water and the organic modified conditions produce comparable results.
modifier of the mobile phase (5 per cent V/V) to prevent
INJECTION VOLUME
crystallisation of salts after completion of the
Where no injection volume is specified in the monograph,
chromatography.
the analyst should select an appropriate volume for their
Mobile phases may contain other components, specific application. The volume chosen is dependent on the
e.g. a counter-ion for ion-pair chromatography or a chiral response of the analyte, the detector used, the efficiency of
selector for chromatography using an achiral stationary phase. the column and the overall performance of the
Detectors chromatographic system. Where a volume is not indicated,
Ultravioletlvisible (UVNis) spectrophotometers, inc1uding 20 flL is usually appropriate; however this should be checked
dio de array detectors, are the most commonly employed for suitability under the local operating conditions.
detectors. Fluorescence spectrophotometers, differential RESOLUTION FACTOR
refractometers, electrochemical detectors, mas s Unless otherwise stated in the monograph, for monographs
spectrometers, light scattering detectors, radioactivity with a stated resolution factor read Resolution (Rs) as referred
detectors or other special detectors may also be used. to in Appendix !II Chromatographic Separation Techniques.
RUNTIME
Method
Where no run time is specified in the monograph, the analyst
Equilibrate the column with the prescribed mobile phase and should select an appropriate run time for their specific
ftow rate, at room temperature or at the temperature application. The run time chosen is dependent on the type of
specified in the monograph, until a stable baseline is test. For example, where a run time is not indicated in a
achieved. Prepare the solution(s) of the substance to ·be Related substances test the analyst should ensure that the run
examined and the reference solution(s) required. time is greater than all known or likely secondary peaksj
The solutions must be free from solid partic1es. similarly in an Assay the run time should be chosen to allow
Criteria for assessing the suitability of the system are the baseline to sta bilis e following the elution of the peak of
described in the chapter on Chromatographic separation interest.
techniques (2.2.46) . The extent to which adjustments of
SECONDARY PEAKS
parameters of the chromatographic system can be made to
Reference may be made to secondary peaks . A secondary peak
is a peak in the chromatogram other than the principal peak
chapter.
and any peak due to intemal standard, solvent or derivatising
Additional points lor monographs 01 the British agents. Peaks identified as being due to the counter-ion
Pharmacopoeia and/or other excipients inc1uding preservatives in the material
The composition and ftow rate of the mobile phase are stated being examined may also be exc1uded.
in the monograph. It is advisable to use as the mobile phase MATERIALS
solvent mixtures that have been de-aerated using a vacuum Solvents and reagents used in the preparation of solutions for
pump or other suitable means of de-aeration that has no examination should be of a quality suitable for use in liquid
effect on the composition of the mixture. chromatography.
In quantitative work, particularly where the use of an intemal
standard is not specified in the monograph, the use of a
fixed-volume loop injector is recommended. In certain
exceptional cases the use of peak heights alone is prescribed
E. Paper Chromatography
in the monograph; where this is the case peak heights should (Ph. Eur. method 2.2.26)
be used irrespective of the symmetry factor.
Ascending Paper Chromatography
The column is usually made of stainless steel and its
dimensions are stated in the monograph. The dimensions are Apparatus The apparatus consists of a glass tank of
stated as (length x intemal diameter) . When the monograph suitable size for the chromatographic paper used, ground at
prescribes the use of a stationary phase designated by a letter, the top to take a c10sely fitting lid. In the top of the tank is a
the relevant stationary phase defined below is intended. device which suspends the chromatographic paper and is
The nominal diameter of the partic1es of the stationary phase capable of being lowered without opening the chamber.
is stated in parentheses irnrnediately following the designating In the bottom of the tank is a dish to contain the mobile
letter. In most cases reference is made to a particular phase into which the paper may be lowered.
commercial brand that has been found to be suitable for the The chromatographic paper consists of suitable filter paper,
purpose, but such statements do not imply that a different cut into strip s of sufficient length and not less than 2.5 cm
but equivalent commercial brand may not be used. wide; the paper is cut so that the mobile phase runs in the
direction of the grain of the paper.
2014 Appendix III F V-A20 1
Method Place in the dish a layer 2.5 cm deep of the

mobile phase prescribed in the monograph. If prescribed in
F. Electrophoresis 1
the monograph, pour the stationary phase between the walls (Ph. Eur. methad 2.2.31)
of the tank and the dish. Close the tank and allow to stand
for 24 h at 20 oC to 25 oc. Maintain the tank at this General PrincipIe
temperature throughout the subsequent procedure. Draw a Under the influence of an electrical field, charged particles
fine pencil line horizontally across the paper 3 cm from one dissolved or dispersed in an electrolyte solution migrate in
end. Using a micro pipette, apply to a spot on the pencilline the direction of the electrode bearing the opposite polarity.
the volume of the solution prescribed in the monograph. In gel electrophoresis, the movements of the particles are
If the total volume to be applied would produce a spot more retarded by interactions with the surrounding gel matrix,
than 10 mm in diameter, apply the solution in portions which acts as a molecular sieve. The opposing interactions of
allowing each to dry before the next application. When more the electrical force and molecular sieving result in differential
than one chromatogram is to be run on the same strip of migration rates according ro sizes, shapes and charges of
paper, space the solutions along the pencil line at points not particles. Because of their different physico-chemical
less than 3 cm aparto Insert the paper into the tank, close the properties, different macromolecules of a mixture will migrate
lid and allow to stand for 1 h 30 mino Lower the paper into at different speeds during electrophoresis and will thus be
the mobile phase and allow elution to proceed for the separated into discrete fractions. Electrophoretic separations
prescribed distance or time. Remove the paper from the tank can be conducted in systems without support phases
and allow to dry in air. Protect the paper from bright light (e.g. free solution separation in capillary electrophoresis) and
during the elution process. in stabilising media such as thin-Iayer plates, film s or gels.
Descending Paper Chromatography Free or Moving Boundary EIectrophoresis
Apparatus The apparatus consists of a glass tank of This method is mainly used for the determination of
suitable size for the chromatographic paper used, ground at mobility, the experimental characteristics being directly
the top to take a closely fitting glass lid. The lid has a central measurable and reproducible. It is chiefly employed with
hole about 1.5 cm in diameter closed by a heavy glass plate substances of high relative molecular mass and low
or a stopper. In the upper part of the tank is suspended a diffusibility. The boundaries are initially located by a physical
solvent trough with a device for holding the chromatographic process such as refractometry or conductimetry. After
paper. On each side of the trough, parallel to and slightly applying a given electric field for an accurately measured
aboye its upper edges, are two glass guide rods to support time, the new boundaries and their respective positions are
the paper in such a manner that no part of it is in contact observed. The operating conditions must be such as to make
with the walls of the tank. The chromatographic paper it possible to determine as many boundaries as there are
consists of suitable filter paper, cut into strips of sufficient components.
length, and of any convenient width berween 2.5 cm and the
length of the trough; the paper is cut so that the mobile Zone EIectrophoresis Using a Supporting Medium
phase runs in the direction of the grain of the paper. This method requires the use of small samples only.
Method Place in the bottom of the tank a layer 2.5 cm The nature of the support, such as paper, agar gel, cellulose
deep of the solvent prescribed in the monograph, close the aceta te, starch, agarose, methacrylamide, mixed gel,
tank and allow to stand for 24 h at 20 oC to 25 oc. introduces a number of additional factors modifying the
Maintain the tan k at this temperature throughout the mobility:
subsequent procedure. Draw a fine pencil line horizontally
a) owing to channelling in the supporting medium; the
across the paper at such a distance from one end that when
apparent distance covered is less than the real distance,
this end is secured in the solvent trough and the remainder
of the paper is hanging freely over the guide rod, the line is a b) sorne su"pporting media are not electrically neutral.
few centimetres below the guide rod and parallel with it. As the medium is a stationary phase it may sometimes
Using a micro-pipette, apply on the pencilline the volume of give rise to a considerable electro-endosmotic flow,
the solution prescribed in the monograph. If the total volume c) any heating due to the joule effect may cause sorne
to be applied would produce a spot more than 10 mm in evaporation of the liquid from the supporting medium
diameter, apply the solution in portions, allowing each to dry which, by capillarity, causes the solution to move from
before the next application. When more than one the ends towards the centre. The ionic strength therefore
chromatogram is to be run on the same strip of paper, space tends to increase gradually.
the solutions along the pencilline at points not less than The rate of migration then depends on four main factors: the
3 cm aparto Insert the paper in the tank, close the lid, and mobility of the charged particle, the electro-endosmotic flow,
allow to stand for 1 h 30 mino Introduce into the solvent the evaporation f1ow, and the field strength. Hence it is
trough, through the hole in the lid, a sufficient quantity of necessary to operate under clearly defined experimental
the mobile phase, close the tank and allow elution to conditions and to use, wherever possible, reference
proceed for the prescribed distance or time. Remove the substances.
paper from the tank and allow to dry in air. The paper An apparalUs for electrophoresis consists of:
should be protected from bright light during the elution
- a generator supplying direct current whose voltage can be
process.
controlled and, preferably, stabilised,
- an electrapharesis chamber. This is usually rectangular and
made of glass or rigid plastic, with rwo separate
compartments, the anodic and the cathodic, containing
1 This chapler has u¡zdergone pharmacopoeial hannonisazion. See chapter
5.8. Phannacopoeial hannonisation (9.18).

V -A202 Appendix III F 2014
the electrolyte solution. In each compartment is immersed tubes into the holders of the upper reservoir and adjust so
an electro de, for example of platinum or graphite. These that the bottom of the tubes are immersed in the buffer
are connected by means of an appropriately isolated solution in the lower reservoir. Carefully fill the tubes with
circuit 10 the corresponding terminal of the power supply the prescribed buffer solution. Prepare the test and reference
to form the anode and the cathode. The level of the liquid solutions containing the prescribed marker dye and make
in the two compartments is kept equal 10 prevent them dense by dissolving in them sucrose R, for example.
siphoning. Apply the solutions 10 the surface of a gel using a different
- The electrophoresis chamber is fitted with an airtight lid tube for each solution. Add the same buffer to the upper
which maintains a moisture-saturated atmosphere during reservoir. Connect the electrodes to the power supply and
operation and reduces evaporation of the solvent. A safety allow electrophoresis 10 proceed at the prescribed
device may be used 10 cut off the power when the lid is temperature and using the prescribed constant voltage or
removed. If the electrical power measured across the strip current. Switch off the power supply when the marker dye
exceeds 10 W, it is preferable to cool the support. has migrated almost into the lower reservoir. Immediately
remove each tube from the apparatus and extrude the gel.
- a support-carrying device:
Locate the position of the bands in the electropherogram as
- Strip electrophoresis. The supporting strip, previously wetted prescribed.
with the same conducting solution and dipped at each end
into an electrode compartment is appropriately tightened SOdiUID Dodecyl Sulfate Polyacrylamide Gel
and fixed on 10 a suitable carrier designed to prevent Electrophoresis (SDS-PAGE)
diffusion of the conducting electrolyte, such as a Scope Polyacrylamide gel electrophoresis is used for the
horizontal frame, inverted-V stand or a uniform surface qualitative characterisation of proteins in biological
with contact points at suitable intervals. preparations, for control of purity and quantitative
- Gel electrophoresis. The device consists essentially of a glass determinations .
plate (for example, a microscope slide) over the whole Purpose Analytical gel electrophoresis is an appropriate
surface of which is deposited a firmly adhering layer of gel method with which 10 identify and to assess the homogeneity
of uniform thickness. The connection between the gel and of proteins in pharmaceutical preparations. The method is
the conducting solution is effected in various ways routinely used for the estimation of protein subunit
according 10 the type of apparatus used. Precautions must molecular masses and for determining the subunit
be taken to avoid condensation of moisture or drying of compositions of purified proteins.
the solid layer.
Ready-to-use gels and reagents are widely available on the
- measuring device or means oi detection. market and can be used instead of those described in this
Method Introduce the electrolyte solution into the text, provided that they give equivalent results and that they
electrode compartments. Place the support suitably meet the validity requirements given below under Validation
impregnated with electrolyte solution in the chamber under of the test.
the conditions prescribed for the type of apparatus used.
Characteristics of polyacrylarnide gels
Locate the starting line and apply the sample. Apply the
electric current for the prescribed time. After the current has The sieving properties of polyacrylamide gels are established
been switched off, remove the support from the chamber, by the three-dimensional network of fibres and pores which is
dry and visualise. formed as the bifunctional bisacrylamide cross-links adjacent
polyacrylamide chains. Polymerisation' is catalysed by a free
Polyacrylamide Rod Gel Electrophoresis radical-generating system composed of ammonium persulfate
and tetramethylethylenediamine.
In polyacrylamide rod gel electrophoresis, the stationary
phase is a gel which is prepared from a mixture of acrylamide As the acrylamide concentration of a gel increases, its
and N,N'-methylenebisacrylamide. Rod gel s are prepared in effective pore size decreases. The effective pore size of a gel is
tubes 7.5 cm long and 0.5 cm in intemal diameter, one operationally defined by its sieving properties; that is, by the
solution being applied to each rod. resistance it imparts 10 the migration of macromolecules.
There are limits on the acrylamide concentrations that can be
Apparatus This consists of two buffer solution reservoirs
used. At high acrylamide concentrations, gels break much
made of suitable material such as poly(methyl methacrylate)
more easily and are difficult to handle. As the pore size of a
and mounted vertically one aboye the other. Each reservoir is
gel decreases, the migration rate of a protein through the gel
fitted with a platinum electrode. The electrodes are
decreases . By adjusting the pore size of a gel, through
connected 10 a power supply allowing operation either at
manipulating the acrylamide concentration, the resolution of
constant current or at constant voltage. The apparatus has in
the method can be optimised for a given protein product.
the base of the upper reservoir a number of holders
Thus, a given gel is physically characterised by its respective
equidistant from the electrode.
composition in acrylamide and bisacrylamide.
Method The solutions should usually be degassed before
In addition to the composition of the gel, the state of the
polymerisation and the gels used immediately after
protein is an important component to the electrophoretic
preparation. Prepare the gel mixture as prescribed and pour
mobility. In the case of proteins, the electrophoretic mobility
into suitable glass tubes, s10ppered at the bottom, to an
is dependent on the pK value of the charged groups and the
equal height in each tube and to about 1 cm from the top,
size of the molecule. It is influenced by the type,
taking care to ensure that no air bubbles are trapped in the
concentration and pH of the buffer, by the temperature and
tubes . Cover the gel mixture with a layer of water R 10
the field strength as well as by the nature of the support
exclude air and allow to seto Gel formation usually takes
about 30 min and is complete when a sharp interface material.
appears between the gel and the water layer. Remove the Denaturing polyacrylarnide gel electrophoresis
water layer. Fill the lower reservoir with the prescribed buffer The method cited as an example is limited to the analysis of
solution and remove the s10ppers from the tubes. Fit the monomeric polypeptides with a mass range of 14 000 10
2014 Appendix nI F v-A203
100000 daltons . Ir is possible to extend this mas s range by Characteristics of discontinuous buffer system gel
various techniques (e.g. gradient gels, particular buffer electrophoresis
system) but those techniques are not discussed in this The most popular electrophoretic method for the
chapter. characterisation of complex mixtures of proteins involves the
Denaturing polyacrylamide gel electrophoresis using sodium use of a discontinuous buffer system consisting of rwo
dodecyl sulfate (SDS-PAGE) is the most common mode of contiguous, but distinct gels: a resolving or separating (lower)
electrophoresis used in assessing the pharmaceutical quality gel and a stacking (upper) gel. The rwo gels are cast with
of protein products and will be the focus of the example different porosities, pH, and ionic strengths. In addition,
method. Typically, analytical electrophoresis of proteins is different mobile ions are used in the gel and electro de
carried out in polyacrylamide gels under conditions that buffers . The buffer discontinuity acts to concentrate large
ensure dissociation of the proteins into their individual volume samples in the stacking gel, resulting in improved
polypeptide subunits and that minimise aggregation. Most resolution. When power is applied, a voltage drop develops
commonly, the strongly anionic detergent sodium dodecyl across the sample solution which drives the proteins into the
sulfate (SDS) is used in combination with heat to dissociate stacking gel. Glycinate ions from the electrode buffer follow
the proteins before they are loaded on the gel. The denatured the proteins into the stacking gel. A moving boundary region
polypeptides bind to SDS, become negatively charged and is rapidly formed with the highly mobile chloride ions in the
exhibit a consistent charge-to-mass ratio regardless of protein front and the relatively slow glycinate ions in the rearo
type. Because the amount of SDS bound is almost always A localised high-voltage gradient forms between the leading
proportional to the molecular mass of the polypeptide and is and trailing ion fronts, causing the SDS-protein complexes to
independent of its sequence, SDS-polypeptide complexes form into a thin zone (stack) and migrate berween the
migrate through polyacrylamide gels with mobilities chloride and glycinate phases. Within broad limits, regardless
dependent on the size of the polypeptide. of the height of the applied sample, aH SDS-proteins
The electrophoretic mobilities of the resultant detergent- condense into a very narrow region and enter the resolving
polypeptide complexes aH assume the same functional gel as a well-defined, thin zone of high protein density.
relationship to their molecular masses. Migration of SDS The large-pore stacking gel do es not retard the migration of
complexes is toward the anode in a predictable manner, with most proteins and serves mainly as an anticonvective
low-molecular-mass complexes migrating fas ter than larger medium. At the interface of the stacking and resolving gels,
ones. The molecular mas s of a protein can therefore be the proteins experience a sharp increase in retardation due to
estimated from its relative mobility in calibrated SDS-PAGE the restrictive pore size of the resolving gel. Once in the
and the occurrence of a single band in such a gel is a resolving gel, proteins continue to be slowed by the sieving of
criterion of purity. the matrix. The glycinate ions overtake the proteins, which
then move in a space of uniformpH formed by the
Modifications to the polypeptide backbone, such as N- or
tris(hydroxymethyl)aminomethane and glycine. Molecular
O-linked glycosylation, however, have a significant impact on
sieving causes the SDS-polypeptide complexes to separate on
the apparent molecular mas s of a protein since SDS does not
bind to a carbohydrate moiety in a manner similar to a the basis of their molecular masses.
polypeptide. Thus, a consistent charge-to-mass ratio is not Preparing vertical discontinuous buffer SDS
maintained. The apparent molecular mass of proteins having polyacrylamide gels
undergone post-translational modifications is not a true Assembling of the gel moulding cassette Clean the rwo
reftection of the mass of the polypeptide chain. glass plates (size: e.g. 10 cm x 8 cm), the
Reducing conditions Polypeptide subunits and three- polytetraftuoroethylene comb, the two spacers and the
dimensional structure is often maintained in proteins by the silicone rubber tubing (diameter e.g. 0.6 mm x 35 cm) with
presence of disulfide bonds. A goal of SDS-PAGE analysis mild detergent and rinse extensively with water. Dry all the
under reducing conditions is to disrupt this structure by items with a paper towel or tissue. Lubricate the spacers and
reducing disulfide bonds . Complete denaturation and the tubing with non-silicone grease. Apply the spacers along
dissociation of proteins by treatment with 2-mercaptoethanol each of the rwo short sides of the glass pI ate 2 mm away
or dithiothreitol (DTT) will result in unfolding of the from the edges and 2 mm away from the long side
polypeptide backbone and subsequent complexation with corresponding to the bottom of the gel. Begin to lay the
SDS . In these conditions, the molecular mas s of the tubing on the glass plate by using one spacer as a guide.
polypeptide subunits can be calculated by linear regression in Carefully twist the tubing at the bottom of the spacer and
the presence of suitable molecular-mas s standards. follow the long side of the glass plate . While holding the
Non-reducing conditions For sorne analyses, complete tubing with one finger along the long side twist again the
dissociation of the protein into subunit peptides is not tubing and lay it on the second short side of the glass pI ate,
desirable. In the absence of treatment with reducing agents using the spacer as a guide. Place the second glass plate in
such as 2-mercaptoethanol or DTT, disulfide covalent bonds perfect alignment and hold the mould together by hand
remain intact, preserving the oligomeric form of the protein. pressure. Apply rwo clamps on each of the two short sides of
Oligomeric SDS-protein complexes migrate more slowly than the mould. Carefully apply four clamps on the longer side of
their SDS-polypeptide subunits. In addition, non-reduced the gel mould thus forming the bottom of the gel mould.
proteins may not be completely saturated with SDS and, Verify that the tubing is running along the edge of the glass
hence, may not bind the detergent in a constant mass ratio. plates and has not been extruded while placing the cJamps.
This makes molecular-mass determinations of these The gel mould is now ready for pouring the gel.
molecules by SDS-PAGE less straighrforward than analyses Preparation of the gel In a discontinuous buffer SDS
of fully denatured polypeptides, since it is necessary that polyacrylamide gel, it is recommended to pour the resolving
both standards and unknown proteins be in similar gel, let the gel set, and then pour the stacking gel since the
configurations for valid comparisons. However, the staining composition of the rwo gels in acrylamide-bisacrylamide,
of a single band in such a gel is a criterion of purity. buffer and pH are different.
V-A204 Appendíx III F 2014
Table 2.2.31.-1. - Preparation of resolving gel
Solution components Component volumes (mL) per gel mould volume of
5mL 50 mL
6 per cent acrylamide
WaterR 2.6 5.3 7.9 10.6 13.2 15.9 21.2 26.5

Acrylamide soIution ll ) 1.0 2.0 3.0 4.0 5.0 6.0 8.0 10.0
1.5 MTris (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 giL APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(') 0.004 0.008 0.012 0.016 0.02 0.024 0.032 0.04
WaterR 2.3 4.6 6.9 9.3 11.5 13.9 18.5 23.2
Acrylamide soIutionO) 1.3 2.7 4.0 5.3 6.7 8.0 10.7 13.3
1.5 M Tris (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 g¡L APS(4) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(') 0.003 0.006 0.009 0.012 0.015 0.018 0.024 0.03
WaterR 1.9 4.0 5.9 7.9 9.9 11.9 15.9 19.8
Acrylamide solutionll ) 1.7 3.3 5.0 6.7 8.3 10.0 13.3 16.7
1.5 M Tris (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 giL APS(' ) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02
WaterR 1.6 3.3 4.9 6.6 8.2 9.9 13.2 16.5

Acrylamide solution(1) 2.0 4.0 6.0 8.0 10.0 12.0 16.0 20.0
1.5 M Tris (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 giL APS(' ) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(') 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02
Water R 1.4 2.7 3.9 5.3 6.6 8.0 10.6 13.8
Acrylamide solution ll ) 2.3 4.6 7.0 9.3 11.6 13.9 18.6 23.2
1.5 M Tris (pH 8.8)(2) 1.2 2.5 3.6 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 giL APS(') 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(') 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02
WaterR 1.1 2.3 3.4 4.6 5.7 6.9 9.2 11.5
Acrylamide solutionll ) 2.5 5.0 7.5 10.0 12.5 15.0 20.0 25.0
1.5 M Tris (pH 8.8)(2) 1.3 2.5 3.8 5.0 6.3 7.5 10.0 12.5
100 giL SDS(3) 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
100 giL APS(') 0.05 0.1 0.15 0.2 0.25 0.3 0.4 0.5
TEMED(5) 0.002 0.004 0.006 0.008 0.01 0.012 0.016 0.02
(1) Acrylamide solution: 30 per cent acrylamidejbisacrylamide(29:1) solution R.

(2) 1.5 M Tris (pH 8.8) : 1.5 M tris-hydrochloride buffer solution pH 8.8 R.
(3) 100 g/L SDS : a 100 g/L soIution of sodium dodecyl sulfate R.
(4) 100 g/L APS: a 100 g/L soIution of ammonium persulfate R. Ammonium persulfate provides the free radicaIs that drive polymerisation of
acrylamide and bisacrylamide. Since ammonium persulfate soIution decomposes slowly, fresh soIutions must be prepared weekly.
(5) TEMED: tetramethylethylenediamine R.
2014 Appendix III F V -A20S
Preparation of the resolving gel In a conical flask, the equipment. Manufacturers of SDS-PAGE equipment
prepare the appropriate volume of solution containing the may provide gel s of different surface area and thickness .
desired concentration of acrylamide for the resolving gel, Electrophoresis running time and currentJvoltage may need
using the values given in Table 2.2.31.-1. Mix the ro vary as described by the manufacturer of the apparatus in
components in the order shown. Where appropriate, before order ro achieve optimum separation. Check that the dye
adding the ammonium persulfate solution and the front is moving into the resolving gel. When the dye is
tetramethylethylenediamine (TEMED), filter the solution if reaching the bottom of the gel, stop the electrophoresis.
necessary under vacuum through a cellulose acetate Remove the gel assembly from the apparatus and separate
membrane (pore diameter 0.45 J.lm); keep the solution under the glass plates. Remove the spacers, cut off and discard the
vacuum by swirling the filtration unit until no more bubbles stacking gel and immediately proceed with staining.
are formed in the solution. Add appropriate amounts of Detection 01 proteins in gels
ammonium persulfate solution and TEMED as indicated in
Coomassie staining is the most common protein staining
Table 2.2 .31.-1, swirl and pour immediately into the gap
method with a detection level of the order of 1 J.lg to 10 J.lg
between the two glass plates of the mould. Leave sufficient
of protein per bando Silver staining is the most sensitive
space for the stacking gel (the length of the teeth of the
method for staining proteins in gel s and a band containing
comb plus 1 cm). Using a tapered glass pipette, carefully
lOng to 100 ng can be detected.
overlay the solution with water-saturated isobutanol. Leave
the gel in a vertical position at room temperature to allow AII of the steps in gel staining are done at room temperature
polymerisation. with gentle shaking (e.g. on an orbital shaker platform) in
any convenient container. Gloves must be worn when
Preparation 01 the stacking gel After polymerisation is
staining gels, since fingerprints will stain.
complete (about 30 min), pour off the isobutanol and wash
the rop of the gel several times with water to remove the Coomassie staining Immerse the gel in a .!arge excess of
isobutanol overlay and any unpolymerised acrylamide. Drain Coomassie staining solutúm R and allow to stand for at least
as much fluid as possible from the top of the gel, and then 1 h. Remove the staining solution.
remove any remaining water with the edge of a paper rowel. Destain the gel with a large excess of destaining solution R .
In a conical fiask, prepare the appropriate volume of solution Change the destaining solution several times, until the
containing the desired concentration of acrylamide, using the stained protein bands are c1early distinguishable on a c1ear
values given in Table 2.2.31.-2 . Mix the components in the background. The more thoroughly the gel is destained, the
order shown. Where appropriate, before adding the smaller is the amount of protein that can be detected by the
ammonium persulfate solution and the TEMED, filter the method . Destaining can be speeded up by inc1uding a few
solution if necessary under vacuum through a cellulose grams of anion-exchange resinor a small sponge in the
acetate membrane (pore diameter: 0.45 J.lm); keep the destaining solution R .
solution under vacuum by swirling the filtration unit until no NOTE: the acid-alcohol solutions used in this procedure do not
more bubbles are fortned in the solution. Add appropriate completely fix proteins in the gel. This can lead to losses of some
amounts of ammonium persulfate solution and TEMED as low-molecular-mass proteins during the staining and destaining of
indicated in Table 2.2.31.-2, swirl and pour immediately into thin gels. Permanent fixation is obtainable by allowing the gel to
the gap between the two glass plates of the mould directly stand in a mixture of 1 volume of trichloroacetic acid R,
onto the surface of the polymerised resolving gel. 4 volumes of methanol R and 5 volumes of water R for 1 h before
Immediately insert a c1ean polytetrafluoroethylene comb into it is immersed in the Coomassie staining solution R.
the stacking gel solution, being careful to avoid trapping air Silver staining Immerse the gel in a large excess of fixing
bubbles. Add more stacking gel solution to fill the spaces of solution R and allow to stand for 1 h. Remove the fixing
the comb completely. Leave the gel in a vertical position and solution, add fresh fixing solution and incubate either for at
allow ro polymerise at room temperature . least 1 h or overnight, if convenient. Discard the fixing
Mounting the gel in the electrophoresis apparatus and solution and wash the gel in a large excess of water R for
electrophoretic separation After polymerisation is 1 h. Soak the gel for 15 min in a 1 per cent V/V solution of
complete (about 30 min), remove the polytetrafluoroethylene glutaraldehyde R. Wash the gel twice for 15 min in a large
comb carefully. Rinse the wells immediately with water or excess of water R . Soak the gel in fresh silver nitra te reagent R
with the SDS-PAGE running buffer R to remove any for 15 min, in darkness. Wash the gel three times for 5 min
unpojymerised acrylamide. If necessary, straighten the teeth in a large excess of water R. Immerse the gel for about 1 min
of the stacking gel with a blunt hypodertnic needle attached in developer solution R until satisfactory staining has been
to a syringe. Remove the c1amps on one short side, carefully obtained. Stop the development by incubation in the blocking
pull out the tubing and replace the c1amps. Proceed similarly solution R for 15 mino Rinse the gel with water R .
on the other short side. Remove the tubing from the bottom Drying 01 stained SDS polyacrylamide gels
part of the gel. Mount the gel in the electrophoresis
Depending on the staining method used, gel s are treated in a
apparatus. Add the electrophoresis buffers to the rop and
slightly different way. For Coomassie staining, after the
bottom reservoirs. Remove any bubbles that become trapped
destaining step, allow the gel to stand in a 100 giL solution
at the bottom of the gel between the glass plates. This is best
of glycerol R for at least 2 h (overnight incubation is possible).
done with a bent hypodertnic needle attached to a syringe.
For silver staining, add to the final rinsing a step of 5 min in
Never pre-run the gel before loading the samples, since this
a 20 giL solution of glycerol R.
will destroy the discontinuity of the buffer systems . Before
loading the sample carefully rinse the slot with SDS-PAGE Immerse two sheets of porous cellulose film in water R and
running buffer R . Prepare the test and reference solutions in incubate for 5 min to 10 mino Place one of the sheets on a
the recommended sample buffer and treat as specified in the drying frame . Carefully lift the gel and place it on the
individual monograph. Apply the appropriate volume of each cellulose film. Remove any trapped air bubbles and pour a
solution to the stacking gel wells. Start the electrophoresis few millilitres of water R around the edges of the gel. Place
using the conditions recommended by the manufacturer of the second sheet on top and remove any trapped air bubbles.
V-A206 Appendix In G 2014
Table 2.2.31.-2. - Preparation of stacking gel

Solution components Component volumes (mL) per gel mould volume oí
1 mL 2mL 3mL 4mL 5mL 6mL 8mL 10mL
Water R 0.68 14 2.1 2.7 3.4 4.1 5.5 6.8
Acrylamide solution{l) 0.17 0.33 0.5 0.67 0.83 1.0 1.3 1.7
1.0 M Tris (pH 6.8){2) 0.13 0.25 0.38 0.5 0.63 0.75 lO 1.25
100 giL SDS(3) 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1
100 giL APS(' I 0.01 0.02 0.03 0.04 0.05 0.06 0.08 0.1
TEMED(51 0.001 0.002 0.003 0.004 0.005 0.006 0.008 0.01
(1) Acrylamide solution: 30 per cent acrylamide/bisacrylamide (29:1) solution R.

(2) lO M Tris (pH 6.8): 1 M tris-hydrochloride buffer solution pH 6.8 R.
(3) 100 giL SDS: a 100 giL solution of sodium dodecyl sulfate R.
(4) 100 giL APS: a 100 giL solution of ammonium persulfate R. Ammonium persulfate provides the free radicals that drive polymerisation of
acrylamide and bisacrylamide. Since ammonium persulfate solution decomposes slowly, fresh solutions must be prepared weekly.
(5) TEMED: tetramethylethylenediamine R.
Complete the assembly of the drying frame. Place in an oven Quantification of impurities
or leave at room temperature until dry. Where the impuriry limit is specified in the individual
Molecular-rnass deterrnination monograph, a reference solution corresponding to that leve!
Molecular mas ses of proteins are determined by comparison of impurity should be prepared by diluting the test solution.
of their mobilities with those of several marker proteins of For example, where the limit is 5 per cent, a reference
known molecular weight. Mixtures of proteins with precisely solution would be a 1:20 dilution of the test solution.
known molecular masses blended for uniform staining are No impurity (any band other than the main band) in the
available for calibrating gels. They are obtainable in various electropherogram obtained with the test solution may be
molecular mas s ranges. Concentrated stock solutions of more intense than the main band obtained with the reference
proteins of known molecular mass are diluted in the solution.
appropriate sample buffer and loaded on the same gel as the Under validated conditions impurities may be quantified by
protein sample ro be studied. normalisation to the main band using an integrating
Immediately after the gel has been run, the position of the densirometer. In this case, the responses must be validated
bromophenol blue tracking dye is marked ro identify the for linearity.
leading edge of the electrophoretic ion front. This can be
done by cutting notches in the edges of the gel or by
inserting a needle soaked in India ink inro the gel at the dye G. Capillary Electrophoresis 1
front. After staining, measure the migration distances ofeach
protein band (markers and unknowns) from the top of the
resolving gel. Divide the migration distance of each protein General PrincipIes
by the distance travelled by the tracking dye . The normalised
Capillary electrophoresis is a physical method of analysis
migration distances so obtained are called the relative
based on the migration, inside a capillary, of charged analytes
mobilities of the proteins (relative to the dye front) and
dissolved in an electrolyte solution, under the infiuence of a
conventionally denoted as R p . Construct a plot of the
direct-current electric fie!d.
logarithm of the relative molecular masses (Mr ) of the protein
standards as a function of the R p values. Note that the The migration velocity of an analyte under an electric field of
graphs are slightly sigmoid. Unknown molecular masses can intensity E, is determined by the electrophoretic mobility of
be estimated by linear regression analysis or interpolation the analyte and the electro-osmotic mobility of the buffer
from the curves of log M r against Rp as long as the values inside the capillary. The electrophoretic mobility of a solute
obtained for the unknown samples are positioned along the (!lep) depends on the characteristics of the solute (electric
linear part of the graph. charge, molecular size and shape) and those of the buffer in
which the migration takes place (type and ionic strength of
Validation 01 the test the electrolyte, pH, viscosity and additives).
The test is not valid unless the proteins of the molecular The e!ectrophoretic ve!ocity (vep) of a solute, assuming a
mass marker are distributed along 80 per cent of the length spherical shape, is given by the equation:
of the gel and over the required separation range
(e.g. the range covering the product and its dimer or the
product and its related impurities) the separation obtained
V ep = fJ. ep x E= (61rqr¡r) x (f)
for the relevant protein bands shows a linear relationship q effective charge of the solute,
between the logarithm of the molecular mass and the R p . 11 viscosity of the electrolyte solution,
Additional validation requirements with respect to the r Sroke's radius of the solute,
solution under test may be specified in individual V applied voltage,
monographs. L rotallength of the capillary.
1 This chapeer has undergone pharmacopoeial harmonisanon. See chapeer

5.8. Pharmacopoeial harmonisation.
2014 Appendix III G V-A207
When an electric field is applied through the capillary filled In practice, other phenomena such as heat dissipation,
with buffer, a ftow of solvent is generated inside the capillary, sample adsorption onto the capillary wall, mismatched
called electro-osmotic ftow. The velocity of the electro- conductivity between sample and buffer, length of the
osmotic ftow depends on the electro-osmotic mobility (fleo) injection plug, detector cell size and unlevelled buffer
which in rum depends on the charge density on the capillary reservoirs can also significantly contribute to band dispersion.
intemal wall and the buffer characteristics. The electro- Separation between 2 bands (expressed as the resolution, R,)
osmotic velocity (veo) is given by the equation: can be obtained by modifying the electrophoretic mobility of
the analytes, the electro-osmotic mobility induced in the
capillary and by increasing the efficiency for the band of each
analyte, according to the equation:
8 = dielectric constant of the buffer,

Rs = VN (J.L epb - J.L epa)
1; = zeta potential of the capillary surface.
4 (JIep + J.L eo)
The velocity of the solute (v) is given by:
v = v ep + Veo fl epa and fl epb electrophoretic mobilities of the 2 analytes

separated,
The electrophoretic mobility of the analyte and the electro- 7lep mean electrophoretic mobility of the
osmotic mobility may act in the same direction or in opposite 2 analytes 7l ep = Y2 ( fl epb + ~lepa)'
directions, depending on the charge of the solute. In normal
capillary electrophoresis, anions will migrate in the opposite APPARATUS
direction to the electro-osmotic ftow and their velocities will An apparatus for capillary electrophoresis is composed of:
be smaller than the electro-osmotic velocity. Cations will ~ a high-voltage, controllable direct-current power supply;
migrate in the same direction as the electro-osmotic ftow and
~ 2 buffer reservoirs, held at the same level, containing the
their velocities will be greater than the electro-osmotic
prescribed ano die and cathodic solutions;
velocity. Under conditions in which there is a fast electro-
osmotic velocity with respect to the electrophoretic velocity of ~ 2 electro de assemblies (the cathode and the anode),
the solutes, both cations and anions can be separated in the immersed in the buffer reservoirs and connected to the
same runo power supply;
The time (t) taken by the solute to migrate the distance (l) ~ a separation capillary (usually made of fused-silica) which,
from the injection end of the capillary to the detection point when used with sorne specific types of detectors, has an
(capillary effective length) is given by the expression: optical viewing window aligned with the detector.
The ends of the capillary are placed in the buffer
lx L reservoirs. The capillary is filled with the solution
t = -- -
Vep + Veo (J.Lep + J.Leo) X V prescribed in the monograph;
~ a suitable injection system;
In general, uncoated fused-silica capillaries aboye pH 3 have ~ a detector able to monitor the amount of substances of
negative charge due to ionised silanol groups in the inner interest passing through a segment of the separation
wall. Consequently, the electro-osmotic ftow is from anode to capillary at a given time; it is usually based on absorption
cathode. The electro-osmotic ftow must remain constant spectrophotometry (UY and visible) or ftuorimetry, but
from run to run if good reproducibility is to be obtained in conductimetric, amperometric or mas s spectrometric
the migration velocity of the solutes . For sorne applications, detection can be useful for specific applications; indirect
it may be necessary to reduce or suppress the electro-osmotic detection is an altemative method used to detect non-UY-
ftow by modifying the inner wall of the capillary or by absorbing and non-ftuorescent compounds;
changing the concentration, composition amjjor pH of the
~ a thermostatic system able to maintain a constant
buffer solution.
temperature inside the capillary is recommended to obtain
After the introduction of the sample into the capillary, each a good separation reproducibility;
analyte ion of the sample migrates within the background ~ a recorder and a suitable integrator or a computer.
electrolyte as an independent zone, according to its
electrophoretic mobility. Zone dispersion, that is the The defrnition of the injection process and its automation are
spreading of each solute band, results from different critical for precise quantitative analysis. Modes of injection
phenomena. Under ideal conditions the sole contribution to include gravity, pressure or vacuum injection and
electrokinetic injection. The amount of each sample
the solute-zone broadening is molecular diffusion of the
solute along the capillary (longitudinal diffusion). In this ideal component introduced electrokinetically depends on its
case the efficiency of the zone, expressed as the number of electrophoretic mobility, leading to possible discrimination
theoretical plates (N), is given by: using this injection mode .
Use the capillary, the buffer solutions, the preconditioning
N = (J.Lep + J.Leo) X V x l method, the sample solution and the migration conditions
2x D x L prescribed in the monograph of the considered substance.
The employed electrolytic solution is filtered to remove
D molecular diffusion coefficient of the solute in the particles and degassed to avoid bubble formation that could
buffer. interfere with the detection system or interrupt the electrical
contact in the capillary during the separation runo A rigorous
rinsing procedure should be developed for each analytical
method to achieve reproducible migration times of the
solutes.
V-A208 Appendix III G 2014
CAPILLARY ZONE ELECTROPHORESIS protein adsorption. In other strategies, the internal wall of the
PrincipIe capillary is coated with a polymer, covalently bonded to the
silica, that prevents interaction between the proteins and the
In capillary zone electrophoresis, analytes are separated in a
negatively charged silica surface. For this purpose, ready-to-
capillary containing only buffer without any anticonvective
use capillaries with coatings consisting of neutral-hydrophilic,
medium. With this technique, separation takes place because
cationic and anionic polymers are available.
the different components of the sample migrate as discrete
bands with different velocities. The velocity of each band ELECTROLYTIC SOLUTION PARAMETERS
depends on the electrophoretic mobility of the solute and the Buffer type and concentration Suitable buffers for
electro-osmotic ftow in the capillary (see General PrincipIes). capillary electrophoresis have an appropriate buffer capacity
Coated capillaries can be used to increase the separation in the pH range of choice and low mobility to minimise
capacity of those substances adsorbing on fused-silica current generation.
surfaces. Matching buffer-ion mobility to solute mobility, whenever
Using this mode of capillary electrophoresis, the analysis of possible, is important for minimising band distortion.
both small (Mr < 2000) and large molecules (2000 < M r < The type of sample solvent used is also important to achieve
100 000) can be accomplished. Due to the high efficiency on-column sample focusing, which increases separation
achieved in capillary zone electrophoresis, separation of efficiency and improves detection.
molecules having only minute differences in their charge-to- An increase in buffer concentration (for a given pH)
mass ratio can be effected. This separation mode also allows decreases electro-osmotic ftow and solute velocity.
the separation of chiral compounds by addition of crural Buffer pH The pH of the buffer can affect separation by
selectors to the separation buffer. modifying the charge of the analyte or additives, and by
Optimisation changing the electro-osmotic ftow. In protein and peptide
Optimisation of the separation is a complex process where separation, changing the pH of the buffer from aboye to
several separation parameters can playa major role. below the isoelectric point (pI) changes the net charge of the
The main factors to be considered in the development of solute from negative to positive. An increase in the buffer pH
separations are instrumental and electrolytic solution generally increases the electro-os moti e ftow.
parameters. Organic solvents Organic modifiers (methanol,
INSTRUMENTAL PARAMETERS
acetonitrile, etc.) may be added to the aqueous buffer to
increase the solubility of the solute or other additives andJor
Voltage A Joule heating plot is useful in optimising the
to affect the degree of ionisation of the sample components .
applied voltage and capillary temperature. Separation time is
The addition of these organic modifiers to the buffer
inversely proportional to applied voltage. However, an
generally causes a decrease in the electro-osmotic ftow .
increase in the voltage used can cause excessive heat
production, giving rise to temperature and, as a result Additives for chirai separations For the separation of
thereof, viscosity gradients in the buffer inside the capillary. optical isomers, a chiral selector is added to the separation
This effect causes band broadening and decreases resolution. buffer. The most commonly used chiral selectors are
cyclodextrins, but crown ethers, polysaccharides and proteins
Polarity Electrode polarity can be normal (anode at the
may also be used. Since crural recognition is governed by the
inlet and cathode at the outlet) and the electro-osmotic ftow
different interactions between the chiral selector and each of
will move toward the cathode. If the electrode polarity is
the enantiomers, the resolution achieved for the chiral
reversed, the electro-osmotic ftow is away from the outlet
compounds depends largely on the type of chiral selector
and only charged analytes with electrophoretic mobilities
used. In this regard, for the development of a given
greater than the electro-osmotic ftow will pass to the outlet.
separation it may be useful to test cyclodextrins having a
Temperature The main effect of temperature is observed different cavity size ((X-, ~-, or y-cyclodextrin) or modified
on buffer viscosity and electrical conductivity, and therefore cyclodextrins with neutral (methyl, ethyl, hydroxyalkyl, etc.)
on migration velocity. In some cases, an increase in capillary or ionisable (aminomethyl, carboxymethyl, sulfobutyl ether,
temperature can cause a conformational change in proteins, etc.) groups. When using modified cyclodextrins, batch-to-
modifying their migration time and the efficiency of the batch variations in the degree of substitution of the
separation. cyclodextrins must be taken into account sine e it will
Capillary The dimensions of the capillary (Iength and inftuence the selectivity. Other factors controlling the
internal diameter) contribute to analysis time, effidency of resolution in chiral separations are concentration of crural
separations and load capacity. Increasing both effective selector, composition and pH of the buffer and temperature .
length and total length can decrease the electric fields The use of organic additives, such as methanol or urea can
(working at constant voltage) wruch increases migration time. also modify the resolution achieved.
For a given buffer and electric field, heat dissipation, and
hence sample band-broadening, depend on the internar Capillary Gel Electrophoresis
diameter of the capillary. The latter also affects the detection Principie
limit, depending on the sample volume injected and the
In capillary gel electrophoresis, separation takes place inside a
detection system employed.
capillary filled with a gel that acts as a molecular sieve.
Since the adsorption of the sample components on the Molecules with similar charge-to-mass ratio s are separated
capillary walllimits efficiency, methods to avoid these according to molecular size sin ce smaller molecules move
interactions should be considered in the development of a more freely through the network of the gel and therefore
separation method. In the specific case of proteins, several migrate faster than larger molecules. Different biological
strategies have been devised to avoid adsorption on the macromolecules (for example, proteins and DNA fragments),
capillary wall. Some of these strategies (use of extreme pH which often have similar charge-to-mass ratio s, can thus be
and adsorption of positively charged buffer additives) only separated according to their molecular mas s by capillary gel
require modification of the buffer composition to prevent electrophoresis.
2014 Appendix III G V -A209
Characteristics oi gels corresponding to their isoelectric point (pI) and the current
2 types of gels are used in capillary electrophoresis: drops to very low values.
permanently coated gels and dynamically coated gels. Mobilisation step If mobilisation is required for detection,
Permanently coated gels, such as cross-linked polyacrylamide, use one of the following methods.
are prepared inside the capillary by polymerisation of the - in the first method, mobilisation is accomplished during
monomers. They are usually bonded to the fused-silica wall the focusing step under the effect of the electro-osmotic
and cannot be removed without destroying the capillary. flow; the electro-osmotic flow must be small enough to
If the gels are used for protein analysis under reducing allow the focusing of the components;
conditions, the separation buffer usually contains sodium
- in the second method, mobilisation is accomplished by
dodecyl sulfate and the samples are denatured by heating in a
applying positive pressure after the focusing step;
mixture of sodium dodecyl sulfate and 2-mercaptoethanol or
dithiothreitol before injection. When non-reducing conditions - in the third method, mobilisation is achieved after the
are used (for example, analysis of an intact antibody), focusing step by adding salts to the cathode reservoir or
2-mercaptoethanol and dithiothreitol are not used. the ano de reservoir (depending on the direction chosen
Separation in cross-linked gels can be optimised by for mobilisation) in order to alter the pH in the capillary
modifying the separation buffer (as indicated in the capillary when the voltage is applied. As the pH is changed, the
zone electrophoresis section) and controlling the gel porosity proteins and ampholytes are mobilised in the direction of
during the gel preparation. For cross-linked polyacrylamide the reservoir which contains the added salts and pass the
gels, the porosity can be modified by changing the detector.
concentration of acrylamide and/or the proportion of cross- The separation achieved, expressed as ilpI, depends on the
linker. As a rule, a decrease in the porosity of the gelleads to pH gradient (dpH/dx), the number of ampholytes having
a decrease in the mobility of the solutes. Due to the rigidity different pI values, the molecular diffusion coefficient (D),
of these gels, only electrokinetic injection can be used. the intensity of the electric field (E) and the variation of the
Dynamically coated gels are hydrophilic polymers, such as electrophoretic mobility of the analyte with the pH
linear polyacrylamide, cellulose derivatives, dextran, etc., (-d¡¡/dpH) :
which can be dissolved in aqueous separation buffers giving
rise to a separation medium that also acts as a molecular D (dpHjdx)
sieve. These separation media are easier to prepare than llpI = 3 x
E (-dll,fdpH)
cross-linked polymers. They can be prepared in a vial and
filled by pressure in a wall-coated capillary (with no electro-
osmotic flow). Replacing the gel before every injection Optimisation
generally improves the separation reproducibility. The main parameters to be considered in the development of
The porosity of the gels can be increased by using polymers separations are:
of higher molecular mass (at a given polymer concentration) Voltage Capillary isoelectric focusing utilises very high
or by decreasing the polymer concentration (for a given electric fields, 300 Vlcm to 1000 V/cm in the focusing step.
polymer molecular mass) . A reduction in the gel porosity
Capillary The electro-osmotic flow must be reduced or
leads to a de crease in the mobility of the solute for the same
suppressed depending on the mobilisation strategy (se e
buffer. Since the dissolution of these polymers in the buffer
aboye). Coated capillaries tend to reduce the electro-osmotic
gives low viscosity solutions, both hydrodynamic and
electrokinetic injection techniques can be used. flow.
Solutions The anode buffer reservoir is filled with a
Capillary Isoelectric Focusing solution with a pH lower than the pI of the most acidic
Principie ampholyte and the cathode reservoir is filled with a solution
with a pH higher than the pI of the most basic ampholyte.
In isoelectric focusing, the molecules migrate under the
Phosphoric acid for the anode and sodium hydroxide for the
influence of the electric field, so long as they are charged, in
cathode are frequently used.
a pH gradient generated by ampholytes having pI values in a
wide range (poly-aminocarboxylic acids), dissolved in the Addition of a polymer, such as methylcellulose, in the
separation buffer. ampholyte solution tends to suppress convective forces (if
any) and electro-osmotic flow by increasing the viscosity.
The three basic steps of isoelectric focusing are loading,
Commercial ampholytes are available covering many pH
focusing and mobilisation.
ranges and may be mixed if necessary to obtain an expanded
Loading step Two methods may be employed: pH range. Broad pH ranges are used to estimate the
- loading in one step: the sample is mixed with ampholytes isoelectric point whereas narrower ranges are employed to
and introduced into the capillary either by pressure or improve accuracy. Calibration can be done by correlating
vacuum; migration time with isoelectric point for a series of protein
- sequential loading: a leading buffer, then the ampholytes, markers.
then the sample mixed with ampholytes, again ampholytes During the focusing step precipitation of proteins at their
alone and finally the terminating buffer are introduced isoelectric point can be prevented, if necessary, using buffer
into the capillary. The volume of the sample must be additives such as glycerol, surfactants, urea or zwitterionic
small enough not to modify the pH gradient. buffers. However, depending on the concentration, urea
Focusing step When the voltage is applied, ampholytes denatures proteins .
migrate toward the cathode or the ano de, according to their
net charge, thus creating a pH gradient from anode (lower Micellar Electroldnetic Chromatography (MEKC)
pH) to cathode (higher pH). During this step the Principie
components to be separated migrate until they reach a pH In micellar electrokinetic chromatography, separation takes
place in an electrolyte solution which contains a surfactant at
V-A21 o Appendix III G 2014
a concentration aboye the critical micellar concentration N number of theoretical plates for one of the
(eme). The solute molecules are distributed between the solutes,
aqueous buffer and the pseudo-stationary phase composed of rJ. se1ectivity,
micelles, according to the partition coefficient of the solute. k'a and k'b retention factors for both solutes, respectively
The technique can therefore be considered as a hybrid of (k'b> k'a)·
electrophoresis and chromatography. It is a technique that
Similar, but not identical, equations give k' and R, values for
can be used for the separation of both neutral and charged
solutes, maintaining the efficiency, speed and instrumental electrically charged solutes.
suitability of capillary e1ectrophoresis. One of the most widely Optimisation
used surfactants in MEKC is the anionic surfactant sodium The main parameters to be considered in the deve10pment of
dodecyl sulfate, although other surfactants, for example separations by MEKC are instrumental and electrolytic
cationic surfactants such as cetyltrimethylammonium salts, solution parameters.
are also used.
INSTRUMENTAL PARAMETERS
The separation mechanism is as follows . At neutral and Voltage Separation time is inversely proportional to
alkaline pH, a strong electro-osmotic flow is generated and applied voltage. However, an increase in voltage can cause
moves the separation buffer ions in the direction of the excessive heat production that gives rise to temperature
cathode. If sodium dodecyl sulfate is employed as the gradients and viscosity gradients of the buffer in the cross-
surfactant, the electrophoretic migration of the anionic section of the capillary. This effect can be significant with
micelle is in the opposite direction, towards the anode. As a high conductivity buffers such as those containing micelles.
result, the overall micelle migration velocity is slowed down Poor heat dissipation causes band broadening and decreases
compared to the bulk flow of the electrolytic solution. In the resolution.
case of neutral solutes, since the analyte can partition
Temperature Variations in capillary temperature affect the
between the micelle and the aqueous buffer, and has no
partition coefficient of the solute between the buffer and the
electrophoretic mobility, the analyte migration velocity will
micelles, the critical miceJlar concentration and the viscosity
depend only on the partition coefficient between the micelle
of the buffer. These parameters contribute to the migration
and the aqueous buffer. In the electropherogram, the peaks
time of the solutes. The use of a good cooling system
corresponding to each uncharged solute are always between
improves the reproducibility of the migration time for the
that of the electro-osmotic flow marker and that of the
solutes.
micelle (the time elapsed between these two peaks is called
the separation window). For electrically charged solutes, the Capillary As in capillary zone electrophoresis, the
migration velocity depends on both the partition coefficient dimensions of the capillary (Iength and intemal diameter)
of the solute between the micelle and the aqueous buffer, contribute to analysis time and efficiency of separations.
and on the electrophoretic mobility of the solute in the Increasing both effective length and total length can decrease
absence of micelle. the electric fields (working at constant voltage), incréase
migration time and improve the separation efficiency.
Since the mechanism in MEKC of neutral and weak1y
The intemal diameter control s heat dissipation (for a given
ionised solutes is essentially chromatographic, migration of
buffer and electric field) and consequently the sample band
the solute and resolution can be rationalised in terms of the
broadening.
retention factor of the solute (k '), also referred to as mass
distribution ratio (DrJ, which is the ratio of the number of ELECTROLYTIC SOLUTION PARAMETERS
moles of solute in the micelle to those in the mobile phase. Surfactant type and concentration The type of
For a neutral compound, k' is given by: surfactant, in the same way as the stationary phase in
chromatography, affects the resolution since it modifies
k' = tR - to =K x Vs
separation se1ectivity. Also, the log k' of a neutral compound
to x (1- -tR)
increases linearJy with the concentration of surfactant in the
VM
mobile phase. Sin ce resolution in MEKC reaches a
t mc
maximum when k' approaches the value of Jtmc/to"
modifYing the concentration of surfactant in the mobile
tR migration time of the solute, phase changes the resolution obtained.
to analysis time of an unretained solute (detennined by Buffer pH Although pH does not modify the partition
injecting an electro-osmotic flow marker which does coefficient of non-ionised solutes, it can modify the electro-
not enter the micelle, for instance methanol), osmotic ftow in uncoated capillaries. A decrease in the buffer
tme micelle migration time (measured by injecting a pH de creases the eJectro-osmotic flow and therefore
micelle marker, such as Sudan lII, which migra tes increases the resolution of the neutral solutes in MEKC,
while continuously associated in the micelle), resulting in a longer analysis time.
K partition coefficient of the solute,
Vs volume of the micellar phase, Organic solvents To improve MEKC separation of
VM volume of the mobile phase. hydrophobic compounds, organic modifiers (methanol,
propanol, acetonitrile, etc.) can be added to the electrolytic
Likewise, the resolution between 2 closely-migrating solutes solution. The addition of these modifiers usually de creases
(R,) is given by: migration time and the se1ectivity of the separation. Since the
addition of organic modifiers affects the critical micellar
1-(~)
concentration, a given surfactant concentration can be used
R =VN x
Ci - 1
x
-.!5L x only within a certain percentage of organic modifier before
the micellisation is inhibited or adversely affected, resulting
s 4 Ci k~ + 1 1 + k~ x (~) in the absence of micelles and, therefore, in the absence of
t mc
partition. The dissociation of micelles in the presence of a
high content of organic solvent does not always mean that
2014 Appendix III G V-A211
the separation will no longer be possible; in sorne cases the

N = 5.54 X t
( Wh
R)2
hydrophobic interaction between the ionic surfactant
monomer and the neutral solutes forms solvophobic
complexes that can be separated electrophoretically. tR migration time or distance along the baseline from the
Additives for chiral separations For the separation of point of injection to the perpendicular dropped from
enantiomers using MEKC, a chiral selector is included in the the maximum of the peak corresponding to the
micellar system, either covalently bound to the surfactant or component,
added to the micellar separation electrolyte. Micelles that Wh = width of the peak at half-height.
have a moiety with chiral discrimination properties include
salts of N-dodecanoyl-L-amino acids, bile salts, etc. Chiral Resolution
resolution can also be achieved using chiral discriminators, The resolution (Rs) between peaks of similar height of
such as cyclodextrins, added to the electrolytic solutions 2 components may be calculated using the expression:
which contain micellised achiral surfactants.
Other additives Several strategies can be carried out to 1.18 X (tR2 - tRl)
Rs = - - - - ' - - - - - ' -
modify selectivity, by adding chemicals to the buffer. Whl + Wh2
The addition of several types of cyclodextrins to the buffer
can also be used to reduce the interaction of hydrophobic tR2 > tRl
solutes with the micelle, thus increasing the selectivity for
this type of compound. tRI and tm migration times or distances along the
The addition of substances able to modify solute-micelle baseline from the point of injection to the
interactions by adsorption on the latter, is used to improve perpendiculars dropped from the maxima of
the selectivity of the separations in MEKC. These additives two adjacent peaks,
may be a second surfactant (ionic or non-ionic) which gives Whl and Wh2 = peak widths at half-height.
rise to mixed micelles or metallic cations which dissolve in When appropriate, the resolution may be calculated by
the micelle and form co-ordination complexes with the measuring the height of the valley (Hv ) between 2 partly
solutes. resolved peaks in a standard preparation and the height of
the smaller peak (Hp ) and calculating the peak-to-valley ratio:
QUANTIFlCATION
Peak areas must be divided by the corresponding migration p
time to give the corrected area in order to: v
- compensate for the shift in migration time from run to
run, thus reducing the variation of the response,
Symmetry factor
- compensate for the different responses of sample
The symmetry factor (As) of a peak may be calculated using
constituents with different migration times.
the expression:
Where an internal standard is used, verify that no peak of the
substance to be examined is masked by that of the internal A _ WO.05
standard. s - 2d
Calculations
From the values obtained, calculate the content of the WO.05 width of the peak at one-twentieth of the peak
component or components being examined. When height,
prescribed, the percentage content of one or more d distance between the perpendicular dropped from
components of the sample to be examined is calculated by the peak maximum and the leading edge of the
determining the corrected area(s) of the peak(s) as a peak at one-twentieth of the peak height.
percentage of the total of the corrected areas of all peaks, Tests for area repeatability (standard deviation of areas or of
excluding those due to solvents or any added reagents the area/migration-time ratio) and for migration time
(normalisation procedure). The use of an automatic repeatability (standard deviation of migration time) are
integration system (integrator or data acquisition and introduced as suitability parameters. Migration time
processing system) is recommended. repeatability provides a test for the suitability of the capillary
washing procedures. An alternative practice to avoid the lack
SYSTEM SUITABILITY of repeatability of the migration time is to use migration time
In order to check the behaviour of the capillary relative to an internal standard.
electrophoresis system, system suitability parameters are A test for the verification of the signal-to-noise ratio for a
used. The choice of these parameters depends on the mode standard preparation (or the determination of the limit of
of capillary electrophoresis used. They are: retention factor quantification) may also be useful for the determination of
(k') (only for micellar electrokinetic chromatography), related substances.
apparent number of theoretical plates (N), symmetry factor
(As) and resolution (R s)' In previous sections, the theoretical Signal-to-noise ratio
expressions for N and Rs have been described, but more The detection limit and quantification limit correspond to
practical equations that allow these parameters to be signal-to-noise ratios of 3 and 10 respectively. The signal-to-
calculated from the electropherograms are given below. noise ratio (SIN) is calculated using the expression:
Apparent number of theoretical plates
S 2H
The apparent number of theoretical plates (N) may be
N h
calculated using the expression:
V -A212 Appendix III H 2014
H height of the peak corresponding to the component (gradient elution of the modifier, pressure (density),
concemed, in the electropherogram obtained with the temperature or flow rate) .
prescribed reference solution, measured from the DETECTORS
maximum of the peak to the extrapolated baseline of Ultraviolet/visible (UVNis) spectrophotometers and flame
the signal observed over a distance equal to twenty ionisation detectors are the most commonly employed
times the width at half-height, detectors. Light scattering detectors, infrared absorption
h range of the background in an electropherogram spectrophotometers, thermal conductivity detectors or other
obtained after injection of a blank, observed over a special detectors may be used.
distan ce equal to twenty times the width at the half-
height of the peak in the electropherogram obtained Method
with the prescribed reference solution and, if possible, Prepare the test solution(s) and the reference solution(s) as
situated equally around the place where this peak prescribed. The solutions must be free from solid partic1es.
would be found.
Criteria for assessing the suitability of the system are
described in the chapter on Chromatographic separation
techniques (2.2.46). The extent to which adjustments of
H. Supercritical Fluid Chromatography parameters of the chromatographic system can be made to
chapter.
Supercritical fluid chromatography (SFC) is a method of
chromatographic separation in which the mobile phase is a
fluid in a supercritical or a subcritical state. The stationary
phase, contained in a column, consists of either finely divided
solid partic1es, such as a silica or porous graphite, a
J. Isoelectric Focusing 1
chemically modified stationary phase, as used in liquid (Ph. Eur. method 2.2.54)
chromatography, or, for capillary columns, a cross-linked
liquid film evenly coated on the walls of the column.
General PrincipIes
SFC is based on mechanisms of adsorption or mass Isoelectric focusing (IEF) is a method of electrophoresis that
distribution. separates proteins according to their isoelectric point.
Separation is carried out in a slab of polyacrylamide or
Apparatus agarose gel that contains a mixture of amphoteric electrolytes
The apparatus usually consists of a cooled pumping system, (ampholytes). When subjected to an electric field, the
an injector, a chromatographic column, contained in an ampholytes migrate in the gel to create a pH gradient.
oven, a detector, a pressure regulator and a data acquisition In sorne cases gels containing an irnmobilised pH gradient,
device (or an integrator or a chart recorder). prepared by incorporating weak acids and bases to specific
regions of the gel network during the preparation of the gel,
PUMPING SYSTEM
are used. When the applied proteins reach the gel fraction
Pumping systems are required to deliver the mobile phase at
that has a pH that is the same as their isoelectric point (pI),
a constant flow rateo Pressure fluctuations are to be
their charge is neutralised and migration ceases. Gradients
minimised, e.g. by passing the pressurised solvent through a
can be made over various ranges of pH, according to the
pulse-damping device. Tubing and connections are capable
mixture of ampholytes chosen.
of withstanding the pressures developed by the pumping
system. Theoretical Aspects
Microprocessor controlled systems are capable of accurately When a protein is at the position of its isoelectric point, it
delivering a mobile phase in either constant or varying has no net charge and cannot be moved in a gel matrix by
conditions, according to a defined programme . In the case of the electric field. It may, however, move from that position
gradient elution, pumping systems which deliver solventes) by diffusion. The pH gradient forces a protein to remain in
from several reservoirs are available and solvent mixing can its isoelectric point position, thus concentrating it; this
be achieved on either the low or high-pressure side of the concentrating effect is called "focusing". Increasing the
pump(s). applied voltage or reducing the sample load result in
INJECTORS improved separation of bands. The applied voltage is limited
Injection may be carried out directly at the head of the by the heat generated, which must be dissipated. The use of
column using a valve. thin gels and an efficient cooling plate controlled by a
STATIONARY PHASES thermostatic circulator prevents the burning of the gel whilst
Stationary phases are contained in columns which have been allowing sharp focusing. The separation is estirnated by
described in the chapters on Liquid chromatography (2.2.29) determining the minimum pI difference (~pI), which is
(packed columns) and Gas chromatography (2.2.28) (capillary necessary to separate 2 neighbouring bands:
columns) . A capillary column has a maximum intemal
diameter (0) of 100 [lm. D (dpHjdx)
~pI =3 x E (-dJ.ljdpH)
MOBILE PHASES
Usually the mobile phase is carbon-dioxide which may
contain a polar modifier such as methanol, 2-propanol or D diffusion coefficient of the protein,
acetonitrile. The composition, pressure (density),
dpH
temperature and flow rate of the prescribed mobile phase pH gradient
may either be constant throughout the whole dx
chromatographic procedure (isocratic, isodense, isothermic 1 This chap¡er has undergone phannacopoeial hannonisation. See chap¡er
elution) or may vary according to a defined programme 5.8. Pharmacopoeial harmonisation.Isoelewic Focusing
2014 Appendix III J V-A213
E intensity of the electric field, in volts Preparation of the gels

per centimetre, Mould The mould (see Figure 2.2.54.-1) is composed of a
dlL glass plate (A) on which a polyester film (B) is placed to
variation of the solute mobility with the pH in facilitate handling of the gel, one or more spacers (C), a
dpH the region close to the pI second glass plate (D) and clamps to hold the structure
1Ogether.
Since D and - dlL for a given protein cannot be altered,
dpH
the separation can be improved by using a narrower pH
range and by increasing the intensity of the electric field.
Resolution between protein bands on an IEF gel prepared
with carrier ampholytes can be quite good. Improvements in
resolution may be achieved by using immobilised pH
gradients where the buffering species, which are analogous 10
carrier ampholytes, are copolymerised within the gel matrix.
Proteins exhibiting pIs differing by as little as 0.02 pH units
may be resolved using a gel prepared with carrier ampholytes
while immobilised pH gradients can resolve proteins differing
by approximately 0.001 pH units .
PRACTICAL ASPECTS
Special attention must be paid to sample characteristics
and/or preparation. Having salt in the sample can be Figure 2.2.54.-1 - Mould
problematic and it is best to prepare the sample, if possible,
in deionised water or 2 per cent ampholytes, using dialysis or 7.5 per cent polyacrylamide gel Dissolve 29.1 g of
gel filtration if necessary. acrylamide R and 0.9 g of methylenebisacrylamide R in 100 mL
The time required for completion of focusing in thin-Iayer of water R. To 2.5 volumes of this solution, add the mixture
polyacrylamide gels is determined by placing a coloured of ampholytes specified in the monograph and dilute to
protein (e.g. haemoglobin) at different positions on the gel 10 volumes with water R. Mix carefuny and degas the
surface and by applying the electric field: the steady state is solution.
reached when an applications give an identical band pattero. Preparation of the mould Place the polyester film on the
In sorne pro1Ocols the completion of the focusing is indicated lower glass plate, apply the spacer, place the second glass
by the time elapsed after the sample application. plate and fit the clamps. Before use, place the solution on a
The IEF gel can be used as an identity test when the magnetic stirrer and add 0.25 volumes of a 100 gIL solution
migration pattero on the gel is compared to a suitable of ammonium persulfate R and 0.25 volumes of
standard preparation and IEF calibration proteins, the IEF tetramethylethylenediamine R. Immediately fin the space
gel can be used as a limit test when the density of a band on between the glass plates of the mould with the solution.
IEF is compared subjectively with the density of bands Method
appearing in a standard preparation, or it can be used as a Dismantle the mould and, making use of the polyester film,
quantitative test when the density is measured using a transfer the gel onto the cooled support, wetted with a few
densitometer or similar instrumentation to determine the millilitres of a suitable liquid, taking care to avoid forming air
relative concentration of protein in the bands subject to bubbles . Prepare the test solutions and reference solutions as
validation. specified in the monograph. Place strips of paper for sample
application, about 10 mm x 5 mm in size, on the gel and
APPARATUS
impregnate each with the prescribed amount of the test and
An apparatus for IEF consists of: reference solutions. AIso apply the prescribed quantity of a
- a contronable generator for constant potential, current and solution of proteins with known isoelectric points as pH
power; potentials of 2500 V have been used and are markers to calibrate the gel. In sorne protocols the gel has
considered optimal under a given set of operating pre-cast slots where a solution of the sample is applied
conditions; a supply of up to 30 W of constant power is instead of using impregnated paper strips. Cut 2 strips of
recommended; paper to the length of the gel and impregnate them with the
- a rigid plastic IEF chamber that contains a cooled plate, electrolyte solutions: acid for the anode and alkaline for the
of suitable material, 10 support the gel; cathode. The compositions of the ano de and cathode
- a plastic cover with platinum electrodes that are solutions are given in the monograph. Apply these paper
connected to the gel by means of paper wicks of suitable wicks 10 each side of the gel several millimetres from the
width, length and thickness, impregnated with solutions of edge. Fit the cover so that the electrodes are in contact with
anodic and cathodic electrolytes. the wicks (respecting the anodic and cathodic poles) . Proceed
with the isoelectric focusing by applying the electrical
Isoelectric Focusing in Polyacrylamide Gels: parameters described in the monograph. Switch off the
Detailed Procedure current when the migration of the mixture of standard
proteins has stabilised. Using forceps, remove the sample
The following method is a detailed descnjJtion of an IEF procedure
application strips and the 2 electrode wicks. Immerse the gel
in thick polyacrylamide slab gels, which is used unless otherwise
in fixing solution for isoelectric focusing in polyacrylamide gel R.
stated in the monograph.
Incubate with gentle shaking at room temperature for
30 mino Drain off the solution and add 200 mL of destaining
V-A214 Appendix III K 2014
solution R. Incubate with shaking for 1 h. Drain the gel, add POINTS TO CONSIDER
coomassie staining solution R. Incubate for 30 mino Destain the Samples can be applied to any area on the gel, but to protect
gel by passive diffusion with destaining solwion R until the the proteins from extreme pH environments samples should
bands are well visualised against a clear background. Locate not be applied close to either electrode. During method
the position and intensity of the bands in the development the analyst can try applying the protein in
electropherogram as prescribed in the monograph. 3 positions on the gel (i.e. middle and both ends);
the pattern of a protein applied at opposite ends of the gel
VARIATIONS TO THE DETAILED PROCEDURE may not be identical.
(SUB]ECT TO VALIDATION)
A phenomenon known as cathodic drift, where the pH
Where reference to the general method on isoelectric gradient decays over time, may occur if a gel is focused too
focusing is made, variations in methodology or procedure long. Although not well understood, electroendoosmosis and
may be made subject to validation. These include: absorption of carbon dioxide may be factors that lead to
- the use of commercially available pre-cast gels and of cathodic drift. Cathodic drift is observed as focused protein
commercial staining and destaining kits, migrating off the cathode end of the gel. Immobilised pH
- the use of immobilised pH gradients, gradients may be used to address this problem.
- the use of rod gels, Efficient cooling (approximately 4 oC) of the bed that the gel
- the use of gel cassettes of different dimensions, including lies on during focusing is important. High field strengths
ultra-thin (0.2 mm) gels, used during isoelectric focusing can lead to overheating and
affect the quality of the focused gel.
- variations in the sample application procedure, including
different sample volumes or the use of sample application
masks or wicks other than paper,
- the use of alternate running conditions, including
variations in the electric field depending on gel dimensions
K. Peptide Mapping 1
and equipment, and the use of fixed migration times (Ph. Eur. method 2.2.55)
rather than subjective interpretation of band stability, Peptide mapping is an identity test for proteins, especially
- the inclusion of a pre-focusing step, those obtained by rDNA technology. It involves the chemical
or enzymatic treatrnent of a protein resulting in the formation
- the use of automated instrumentation,
of peptide fragments followed by separation and
- the use of agarose gels. identification of these fragments in a reproducible manner.
It is a powerful test that is capable of identifying almost any
VALIDATION OF ISO-ELECTRIC FOCUSING single amino acid changes resulting from events such as
PROCEDURES errors in the reading of complementary DNA (cDNA)
Where alternative methods to the detailed procedure are sequences or point mutations. Peptide mapping is a
employed they must be validated. The following criteria may comparative procedure because the information obtained,
be used to validate the separation: compared to a reference substance similarly treated, confirms
- formation of a stable pH gradient of desired the primary structure of the protein, is capable of detecting
characteristics, assessed for example using coloured pH whether alterations in structure have occurred, and
markers of known isoelectric points, demonstrates process consistency and genetic stability. Each
- comparison with the electropherogram provided with the protein presents unique characteristics which must be well
chemical reference substance for the preparation to be understood so that the scientific and analytical approaches
examined, permit validated developmentof a peptide map that provides
sufficient specificity.
- any other validation criteria as prescribed in the
monograph. This chapter provides detailed assistance in the application of
peptide mapping and its validation to characterise the desired
SPECIFIED VARIATIONS TO THE GENERAL protein, to evaluate the stability of the expression construct of
METHOD cells used for recombinant DNA products and to evaluate
the consistency of the, overall process, to assess product
Variations to the general method required for the analysis of
stability as well as to ensure the identity of the protein, or to
specific substances may be specified in detail in monographs.
detect the presence of protein variant.
These include:
Peptide mapping is not a general method, but involves
- the addition of urea in the gel (3 M concentration is often
developing specific maps for each unique protein. Although
satisfactory to keep protein in solution but up to 8 M can
the technology is evolving rapidly, there are certain methods
be used): some proteins precipitate at their isoelectric
that are generally accepted. Variations of these methods will
point; in this case, urea is included in the gel formulation
be indicated, when appropriate, in specific monagraphs.
to keep the protein in solution; if urea is used, only fresh
solutions should be used to prevent carbamylation of the A peptide map may be viewed as a fingerprint af a pratein
protein; and is the end product af several chemical processes that
pravide a camprehensive understanding af the protein being
- the use of alternative staining methods;
analysed. 4 principal steps are necessary far the development
- the use of gel additives such as non-ionic detergents of the pracedure: isolatian and purification of the protein, if
(e.g. octylglucoside) or zwitterionic detergents (e.g., the protein is part of a formulatian; selective cleavage af the
CHAPS or CHAPSO), and the addition of ampholyte to peptide bands; chromatographic separatian of the peptides;
the sample, to prevent proteins from aggregating or and analysis and identification of the peptides. A test sample
precipitating.
1 This chapter has undergone phannacopoeial hannonisation. See chapter
5. 8. Pharrnacopoeial harrnonisation.
2014 Appendix III K V-A215
TabIe 2.2.55.-1. - Examples of cleavage agents

Type Agent Specificity
Enzymatic Trypsin (EC 3.4.21.4) e-terminal side of Arg and Lys
Chymotrypsin (EC 3.4.21.1) e-terminal side of hydrophobic residues (e.g. Leu, Met, Ala,
aromatics)
Pepsin (EC 3.4.23.1 and 2) Non'specific digest
Lysyl endopeptidase (Lys-C endopeptidase) (EC 3.4.21.50) e-terminal side of Lys
Glutamyl endopeptidase (from S. aureus strain V8) (EC e -terminal side of Glu and Asp
3.4.21.19)
Peptidyl-Asp metallo-endopeptidase (endoproteinase Asp-N) N-terminal si de of Asp
Clostripain (EC 3.4.22.8) e -terminal side of Arg
Chemical Cyanogen bromide e-terminal si de of Met
2-Nitro-5-thio-cyanobenzoic acid N-terminal side of Cys
O-Iodosobenzoic acid e-terminal side of Trp and Tyr
Dilute acid Asp and Pro
BNPS-skatole Trp
is digested and assayed in paralIeI with a reference substance. the protein from excipients and stabilisers used in
Complete eleavage of peptide bonds is more likely to occur formulation of the product, if these interfere with the
when enzymes such as endoproteases (e.g., trypsin) are used, mapping procedure. Physical procedures used for
instead of chemical eleavage reagents. A map must contain pretreatment can inelude ultrafiltration, column
enough peptides to be meaningful. On the other hand, if chroma1Ography and Iyophilization. Other pretreatments,
there are too many fragments, the map might lose its such as the addition of chaotropic agents (e.g. urea) can be
specificity because many proteins will then have the same used to unfold the protein prior 10 mapping. To allow the
profiles. enzyme to have full access to eleavage sites and permit sorne
unfolding of the protein, it is often necessary to reduce and
Isolation and Purification alkylate the disulfide bonds prior 10 digestion.
Isolation and purification are necessary for analysis of bulk Digestion with trypsin can introduce ambiguities in the
drugs or dosage forms containing interfering excipients and peptide map due 10 side reactions occurring during the
carrier proteins and, when required, will be specified in the digestion reaction, suchas non-specific eleavage,
monograph. Quantitative recovery of protein from the dosage deamidation, disulfide isomerisation, oxidation of methionine
form must be validated. residues, or formation of pyroglutamic groups created from
the deamidation of glutamine at the N-terminal side of a
Selective Cleavage of Peptide Bonds peptide. Furthermore, peaks may be produced by
The selection of the approach used for the eleavage of autohydrolysis of trypsin. Their intensities depend on the
peptide bonds will depend on the protein under test. This ratio of trypsin 10 protein. To avoid autohydrolysis, solutions
selection process involves determination of the type of of proteases may be prepared at a pH that is not optimal
eleavage to be employed, enzymatic or chemical, and the (e.g. at pH 5 for trypsin), which would mean that the
type of eleavage agent within the chosen category. SeveraI enzyme would not become active until diluted with the digest
c1eavage agents and their specificity are shown in buffer.
Table 2.2.55 .-1. This list is not all-inc1usive and will be Establishment of optimal digestion conditions Fac10rs
expanded as other c1eavage agents are identified. that affect the completeness and effectiveness of digestion of
Pretreatment of sample Depending on the size or the proteins are those that could affect any chemical or
configuration of the protein, different approaches in the enzymatic reactions.
pretreatment of samples can be used. If trypsin is used as a pH o/ the reaction milieu The pH of the digestion
c1eavage agent for proteins with a molecular mas s greater mixture is empirically determined to ensure the optimisation
than 100 000 Da, Iysine residues must be protected by of the performance of the given eleavage agent. For example,
citraconyIation or maleylation; otherwise, too many peptides when using cyanogen bromide as a eleavage agent, a highly
will be generated. acidic environment (e.g. pH 2, formic acid) is necessary;
Pretreatment of the cleavage agent Pretreatment of however, when using trypsin as a c1eavage agent, a slightly
c1eavage agents, especialIy enzymatic agents, might be alkaline environment (pH 8) is optima!. As a general rule,
necessary for purification purposes to ensure reproducibility the pH of the reaction milieu must not alter the chemical
of the map. For example, trypsin used as a c1eavage agent integrity of the protein during the digestion and must not
will have to be treated with tosyl-L-phenylalanine change during the course of the fragmentation reaction.
chloromethyl ketone 10 inactivate chymotrypsin. Other Temperature A temperature between 25 oC and 37 oC is
methods, such as purification of trypsin by high performance adequate for most digestions. The temperature used is
liquid chroma1Ography (HPLC) or immobilisation of enzyme intended to minimise chemical side reactions . The type of
on a gel support, have been successfully used when only a protein under test will dicta te the temperature of the reaction
smalI amount of protein is available. milieu, because sorne proteins are more susceptible to
Pretreatment of the protein Under certain conditions, it denaturation as the temperature of the reaction increases.
might be necessary 10 concentrate the sample or 10 separate For example, digestion of recombinant bovine somatropin is
V-A216 Appendix In K 2014
conducted at 4 oc, because at higher temperatures it will 0.1 per cent triftuoroacetic acid is added. If necessary, add
precipitate duting digestion. propyl alcohol or isopropyl alcohol to solubilise the digest
Time If sufficient sample is available, a time course study components, provided that the addition does not unduly
is considered in order to determine the optimum time to increase the viscosity of the components.
obtain a reproducible map and avoid incomplete digestion. Mobile phase Buffered mobile phases containing
Time of digestion varies from 2 h to 30 h. The reaction is phosphate are used to provide sorne ftexibility in the
stopped by the addition of an acid which does not interfere selection of pH conditions, sin ce shifts of pH in the 3.0-5 .0
in the map or by freezing. range enhance the separation of peptides containing acidic
Amount 01 cleavage agent used Although excessive residues (e.g. glutamic and aspartic acids). Sodium or
amounts of eleavage agent are used to accomplish a potassium phosphates, arnmonium acetate, phosphoric acid
reasonably rapid digestion time (i.e. 6-20 hours), the amount at a pH between 2 and 7 (or higher for polymer-based
of eleavage agent is minimised to avoid its contribution to supports) have also been used with acetonitrile gradients.
the chromatographic map pattern. A protein to protease ratio Acetonitrile containing trifiuoroacetic acid is used quite
between 20: 1 and 200: 1 is generaIly used. It is often.
recommended that the eleavage agent is added in 2 or more Gradient Gradients can be linear, nonlinear, or inelude
stages to optimise eleavage. Nonetheless, the final reaction step functions. A shaIlow gradient is recommended in order
volume remains smaIl enough to facilitate the next step in to separate complex mixtures. Gradients are optimised to
peptide mapping, the separation step. To sort out digestion provide elear resolution of 1 or 2 peaks that wilI become
artifacts that might interfere with the subsequent analysis, a "marker" peaks for the test.
blank determination is performed, using a digestion control Isocratic elution Isocratic HPLC systems using a single
with aIl the reagents, except the test protein. mobile phase are used on the basis of their convenience of
use and improved detector responses. Optimal composition
Chromatographic Separation of a mobile phase to obtain elear resolution of each peak is
Many techniques are used to separate peptides for mapping. sometimes difficult to establish. Mobile phases for which
The selection of a technique depends on the protein being slight changes in component ratio s or in pH significantly
mapped. Techniques that have been successfuIly used for affect retention times of peaks in peptide maps must not be
separation of peptides are shown in Table 2.2.55-2. In this used in isocratic HPLC systems .
section, a most widely used reversed-phase HPLC method is Other parameters Temperature control of the column is
described as one of the procedures of chromatographic usuaIly necessary to achieve good reproducibility. The ftow
separation. rates for the mobile phases range from 0.1-2.0 mUmin, and
The purity of solvents and mobile phases is a critical factor in the detection of peptides is performed with a UV detector at
HPLC separation. HPLC-grade solvents and water that are 200-230 nm. Other methods of detection have been used
commerciaIly available, are recommended for reversed-phase (e.g. post-column derivatisation), but they are not as robust
HPLC. Dissolved gases present a problem in gradient or versatile as UV detection.
systems where the solubility of the gas in a solvent may be Validation This section provides an experimental means
less in a mixture than in a single solvent. Vacuum degassing for measuring the overaIl performance of the test method.
and agitation by sonication are often used as useful degassing The acceptance criteria for system suitability depend on the
procedures. When solid partieles in the solvents are drawn identification of critical test parameters that affect data
into the HPLC system, they can damage the sealing of pump interpretation and acceptance. These critical parameters are
val ves or elog the top of the chromatographic column. Both also criteria that monitor peptide digestion and peptide
pre- and post-pump filtration is also recommended. analysis. An indicator that the desired digestion endpoint has
been achieved is shown by comparison with a reference
Table 2.2.55-2. - Techniques used for the separation of
standard, which is treated in the same mauner as the test
peptides
protein. The use of a reference substance in paraIlel with the
Reversed·phase high performance liquid chromatography (HPLC) test protein is critical in the development and establishment
lon-exchange chromatography (lEC) of system suitability limits. In addition, a chromatogram is
ineluded with the reference substance for additional
Hydrophobic interaction chromatography (HIC)
comparison purposes. Other indicators may inelude visual
Polyacrylamide gel electrophoresis (PAGE), non-denaturating inspection of protein or peptide solubility, the absence of
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) intact protein, or measurement of responses of a digestion-
dependent peptide . The critical system suitability parameters
Capillary electrophoresis (CE)
for peptide analysis wil! depend on the particular mode of
Papér chromatography·high voltage (PCHV) peptide separation and detection and on the data analysis
High voltage·paper electrophoresis (HVPE) requirements.
When peptide mapping is used as an identification test, the
system suitability requirements for the identified peptides
Chromatographic column The selection of a
cover selectivity and precision. In this case, as weIl as when
chromatographic column is empiricaIly determined for each
identification of variant protein is done, the identification of
protein. Columns with 10 nm or 30 nm pore size with silica
the primary structure of the peptide fragments in the peptide
support can give optimal separation. For smaIler peptides,
map provides both a verification of the known primary
oetylsilyl sitúa gel for ehromatography R (3-10 Ilm) and
structure and the identification of protein variants by
oetadecylsilyl siliea gelfor ehromatography R (3-10 Ilm) column
comparison with the peptide map of the reference substance
packings are more efficient than butylsilyl siliea gel for
for the specified protein. The use of a digested reference
ehromatography R (5-10 !lm).
substance for a given protein in the determination of peptide
Solvent The most commonly used solvent is water with resolution is the method of choice. For an analysis of a
acetonitrile as the organic modifier to which not more than
2014 Appendix III L V-A21 7
variant protein, a characterised mixture of a variant and a The possibility of auro-hydrolysis of trypsin is monirored by
reference substance can be used, especially if the variant producing a blank peptide map, that is, the peptide map
peptide is located in a less-resolved region of the map. obtained when a blank solution is treated with trypsin.
The index of pattern consistency can be simply the number The minimum requirement for the qualification of peptide
of major peptides detected. Peptide pattern consistency can mapping is an approved test procedure that includes system
be best defined by the resolution of peptide peaks. suitability as a test control. In general, early in the regulatory
Chromatographic parameters, such as peak-to-peak process, qualification of peptide mapping for a protein is
resolution, maximum peak width, peak area, peak tailing sufficient. As the regulatory approval process for the protein
factors, and column efficiency, may be used to define peptide progresses, additional qualifications of the test can include a
resolution. Depending on the protein under test and the parrial validation of the analytical procedure ro provide
method of separation used, single peptide or multiple peptide assurance that the method will perform as intended in the
resolution requirements may be necessary. development of a peptide map for the specified protein.
The replicate analysis of the digest of the reference substance
for the protein under test yields mea sures of precision and Analysis and Identification of Peptides
quantitative recovery. Recovery of the identified peptides is This section gives guidance on the use 01 peptide mapping during
generally ascertained by the use of internal or external development in support 01 regulatory applications.
peptide standards . The precision is expressed as the relative The use of a peptide map as a qualitative tool does not
standard deviation (RSD) . Differences in the recovery and require the complete characterisation of the individual
precision of the identified peptides are to be expected; peptide peaks. However, validation of peptide mapping in
therefore, the system suitability Iimits will have to be support of regulatory applications requires rigorous
established for both the recovery and the precision of the characterisation of each of the individual peaks in the peptide
identified peptides. These Iimirs are unique for a given map . Methods to characterise peaks range from N-terminal
protein and will be specified in the individual monograph. sequencing of each peak followed by amino acid analysis ro
Visual comparison of the relative retentions, the peak the use of mass spectroscopy (MS).
responses (the peak area or the peak height), the number of For characterisation purposes, when N-terminal sequencing
peaks, and the overall elution pattern is completed initially. and amino acids analysis are used, the analytical separation is
It is then complemented and supported by mathematical scaled up. Since scale-up might affect the resolution of
analysis of the peak response ratio s and by the peptide peaks, it is necessary, using empirical data, ro assure
chromarographic profile of al: 1 (VN) mixture of sample that there is no loss of resolution due ro scale-up. Eluates
and reference substance digesto If all peaks in the sample corresponding to specific peptide peaks are collected,
digest and in the reference substance digest have the same vacuum-concentrated, and chromatographed again, if
relative retentions and peak response ratios, then the identity necessary. Amino acid analysis of fragrnents may be Iimited
of the sample under test is confirmed. by the peptide size. If the N-terminus is blocked, ir may need
If peaks that initially eluted with significantly different relative ro be cleared before sequencing. e-terminal sequencing of
retentions are then observed as single peaks in the 1:1 proteins in combination with carboxypeptidase and matrix-
mixture, the initial difference would be an indication of assisted laser desorption ionisation coupled to time-of-flight
system variability. However, if separate peaks are observed in analyser (MALDI-TOF) can also be used for characterisation
the 1:1 mixture, this would be evidence of the purposes.
nonequivalence of the peptides in each peak. If a peak in the The use of MS for characterisation of peptide fragrnents is by
1: 1 mixture is significantly broader than the corresponding direct infusion of isolated peptides or by the use of on-Iine
peak in the sample and reference substance digest, it may LC-MS for structure analysis. In general, it includes
indicate the presence of different peptides. The use of electrospray and MALDI-TOF-MS, as well as fast-atom
computer-aided pattern recognition software for the analysis bombardment (FAB) . Tandem MS has also been used ro
of peptide mapping data has been proposed and applied, but sequence a modified protein and ro determine the type of
issues related to the validation of the computer software amino acid modification that has occurred. The comparison
preclude its use in a compendial test in the near future. of mass spectra of the digests before and after reduction
Other quromated approaches have been used that employ provides a method to assign the disulfide bonds ro the
mathematical formulas, models, and pattern recognition. various sulfydryl-containing peptides.
Such approaches are, for example, the automated If regions of the primary structure are not clearly
identification of compounds by IR spectroscopy and the demonstrated by the peptide map, it might be necessary to
application of diode-array UV spectral analysis for develop a secondary peptide map. The goal of a validated
identification of peptides. These methods have Iimitations method of characterisation of a protein through peptide
due ro inadequate resolutions, co-elution of fragrnents, or mapping is ro reconcile and account for at least 95 per cent
absolute peak response differences between reference of the theoretical composition of the protein structure.
substance and sample digest fragrnents.
The numerical comparison of the peak retention times and
peak areas or peak heights can be done for a selected group
of relevant peaks that have been correctly identified in the L. Amino Acid Analysis 1
peptide maps. Peak areas can be calculated using 1 peak (Ph. Eur. mechod 2.2.56)
showing relatively small variation as an internal reference, Amino acid analysis refers to the methodology used to
keeping in mind that peak area integration is sensitive ro determine the amino acid composition or content of proteins,
baseline variation and Iikely ro introduce error in the analysis. peptides, and other pharmaceutical preparations. Proteins
Alternatively, the percentage of each peptide peak height and peptides are macromolecules consisting of covalently
relative ro the sum of all peak heights can be calculated for
the sample under test. The percentage is then compared ro J This chapter has undergone phamzacopoeial harJ11onisaúon. See chapter
that of the corresponding peak of the reference substance. 5.8. Pharmacopoeial harmonisationAmino Acid Analysis
V -A218 Appendix III L 2014
bonded amino acid residues organised as a linear polymer. Reference Material

The sequence of the amino acids in a protein or peptide Acceptable amino acid standards are commercially available
determines the properties of the molecuJe. Proteins are for amino acid analysis and typically consist of an aqueous
considered large molecules that commonly exist as folded mixture of amino acids. When determining amino acid
structures with a specific conformation, while peptides are composition, protein or peptide standards are analysed with
smaller and may consist of only a few amino acids. Amino the test material as a control to demonstrate the integrity of
acid analysis can be used to quantifY proteins and peptides, the entire procedure. Highly purified bovine serum albumin
to determine the identity of proteins or peptides based on has been used as a protein standard for this purpose .
their amino acid composition, to support protein and peptide
structure analysis, to evaluate fragmentation strategies for Calibration of Instrumentation
peptide mapping, and to detect atypical amino acids that Calibration of amino acid analysis instrumentation typically
might be present in a protein or peptide. It is necessary to involves analysing the amino acid standard, which consists of
hydrolyse a proteinlpeptide to its individual amino acid a mixture of amino acids at a number of concentrations, to
constituents before amino acid analysis. Following determine the response factor and range of analysis for each
proteinlpeptide hydrolysis, the amino acid analysis procedure amino acid. The concentration of each amino acid in the
can be the same as that practiced for free amino acids in standard is known. In the calibration procedure, the analyst
other pharmaceutical preparations. The amino acid dilutes the amino acid standard to several different analyte
constituents of the test sample are typically derivatised for
levels within the expected linear range of the amino acid
analysis.
analysis technique. Then, replicates at each of the different
analyte levels can be analysed. Peak areas obtained for each
Apparatus
amino acid are plotted versus the known concentration for
Methods used for amino acid analysis are usuaUy based on a each of the amino acids in the standard dilution. These
chromatographic separation of the amino acids present in the results wiU allow the analyst to determine the range of amino
test sample. Current techniques take advantage of the acid concentrations where the peak area of a given amino
automated chromatographic instrumentation designed for acid is an approximately linear function of the amino acid
analytical methodologies. An amino acid analysis instrument concentration. Ir is important that the analyst prepare the
wiU typicaUy be a low-pressure or high-pressure liquid samples for amino acid analysis so that they are within the
chromatograph capable of generating mobile phase gradients analyticallimits (e.g., linear working range) of the technique
that separate the amino acid analytes on a chromatographic employed in order to obtain accurate and repeatable results .
column. The instrument must have post-column
4 to 6 amino acid standard levels are analysed to determine a
derivatisation capability, unless the sample is analysed using
response factor for each amino acid. The response factor is
precolumn derivatisation. The detector is usually an
calculated as the average peak area or peak height per
ultraviolet/visible or ftuorescence detector depending on the
nanomole of amino acid present in the standard.
derivatisation method used. A recording device (e.g.,
A calibration file consisting of the response factor for each
integrator) is used for transforming the analogue signal from
amino acid is prepared and used to calculate the
the detector and for quantitation. Ir is preferred that
concentration of each amino acid present in the test sample.
instrumentation be dedicated particularly for amino acid
This calculation involves dividing the peak area
analysis.
corresponding to a given amino acid by the response factor
General Precautions for that amino acid to give the nanomoles of the amino acid.
For routine analysis, a single-point calibration may be
Background contamination is always a concem for the sufficient; however, the calibration file is updated frequently
analyst in performing amino acid analysis. High purity and tested by the analysis of analytical controls to ensure its
reagents are necessary (e.g., low purity hydrochloric acid can integrity.
contribute to glycine contamination). Analytical reagents are
changed routinely every few weeks using only high-pressure Repeatability
Iiquid chromatography (HPLC) grade solvents. Potential
Consistent high quality amino acid analysis results from an
microbial contamination and foreign material that might be
analyticallaboratory require attention to the repeatability of
present in the solvents are reduced by filtering solvents before
the assay. During analysis of the chromatographic separation
use, keeping solvent reservoirs covered, and not placing
of the amino acids or their derivatives, numerous peaks can
amino acid analysis instrumentation in direct sunlight.
be observed on the chromatogram that correspond to the
Laboratory practices can determine the quality of the amino amino acids. The large number of peaks makes it necessary
acid analysis. Place the instrumentation in a low traffic area to have an amino acid analysis system that can repeatedly
of the laboratory. Keep the laboratory cJean. Clean and identifY the peaks based on retention time and integrate the
calibrate pipets according to a maintenance schedule. Keep peak are as for quantitation. A typical repeatability evaluation
pipet tips in a covered box; the analysts may not handle pipet involves preparing a standard amino acid solution and
tips with their hands. The analysts may wear powder-free analysing many replicates (e.g., 6 analyses or more) of the
latex or equivalent gloves. Limit the number of times a test same standard solution. The relative standard deviation
sample vial is opened and cJosed because dust can contribute (RSD) is determined for the retention time and integrated
to elevated levels of glycine, serine, and alanine. peak area of each amino acid. An evaluation of the
A well-maintained instrument is necessary for acceptable repeatability is expanded to incJude multiple assays
amino acid analysis results. If the instrument is used on a conducted over several days by different analysts. Multiple
routine basis, it is to be checked daily for leaks, detector and assays incJude the preparation of standard dilutions from
lamp stability, and the ability of the column to maintain starting materials to determine the variation due to sample
resolution of the individual amino acids. Clean or replace all handling. The amino acid composition of a standard protein
instrument filters and other maintenance items on a routine (e.g., bovine serum albumin) ís often analysed as part of the
schedule. repeatability evaluatíon. By evaluating the replicate variation
2014 Appendix III L V -A219
(i.e., RSD), the laboratory can establish analyticallimits to hydrolysis tubes are rinsed with high-purity water followed by
ensure that the analyses from the laboratory are under a rinse with HPLC grade methanol, dried overnight in an
control. It is desirable to establish the lowest practical oven, and stored covered until use. Alternatively, pyrolysis of
variation limits to ensure the best results. Areas to focus on clean glassware at 500 oC for 4 h may also be used to
to lower the variability of the amino acid analysis include eliminate contamination from hydrolysis tubes. Adequate
sample preparation, high background spectral interference disposable laboratory material can also be used.
due 10 quality of reagents and/or laboratory practices, Acid hydrolysis is the most common method for hydrolysing
instrument performance and maintenance, data analysis and a protein sample before amino acid analysis. The acid
interpretation, and analyst performance and habits. hydrolysis technique can contribute to the variation of the
All parameters involved are fully investigated in the scope of analysis due to complete or partial destruction of several
the validation work. amino acids: tryptophan is destroyedi serine and threonine
are partially destroyed; methionine might undergo oxidation;
Sample Preparation
and cysteine is typically recovered as cystine (but cystine
Accurate results from amino acid analysis require purified recovery is usually poor because of partial destruction or
protein and peptide samples. Buffer components (e.g., salts, reduction to cysteine) . Application of adequate vacuum (less
urea, detergents) can interfere with the amino acid analysis than 200 ¡.tm of mercury or 26.7 Pa) or introduction of an
and are removed from the sample before analysis. Methods inert gas (argon) in the headspace of the reaction vessel can
that utilise post-colurnn derivatisation of the amino acids are reduce the level of oxidative destruction. In peptide bonds
generally not affected by buffer components 10 the extent involving isoleucine and valine the amido bonds of De-De,
seen with pre-column derivatisation methods. It is desirable Val-Val, De-Val, and Val-De are partially cleaved;
to limit the number of sample manipulations 10 reduce and asparagine and glutamine are deamidated, resulting in
potential background contamination, to improve analyte aspartic acid and glutamic acid, respectively. The loss of
recovery, and to reduce labour. Common techniques used to tryp1Ophan, asparagine, and glutamine during an acid
remove buffer components from protein samples include the hydrolysis limits quantitation 10 17 amino acids. Sorne of the
following methods: (1) injecting the protein sample onto a hydrolysis techniques described are used to address these
reversed-phase HPLC system, removing the protein with a concerns. Sorne of the hydrolysis techniques described (Le.,
volatile solvent containing a sufficient organic component, Methods 4-11) may cause modifications 10 other amino
and drying the sample in a vacuum centrifuge; (2) dialysis acids. Therefore, the benefits of using a given hydrolysis
against a volatile buffer or water; (3) centrifugal ultrafiltration technique are weighed against the concerns with the
for buffer replacement with a volatile buffer or water; technique and are tested adequately before employing a
(4) precipitating the protein from the buffer using an organic method other than acid hydrolysis.
solvent (e.g., acetone); (5) gel filtration. A time-course study (Le., amino acid analysis at acid
Internal Standards hydrolysis times of 24 h, 48 h and 72 h) is often employed to
analyse the starting concentration of amino acids that are
It is recommended that an internal standard be used 10 partially destroyed or slow to cleave. By plotting the observed
monitor physical and chemical losses and variations during concentration of labile amino acids (e.g., serine and
amino acid analysis. An accurately known amount of internal threonine) versus hydrolysis time, the line can be
standard can be added to a protein solution prior to
extrapolated to the origin to determine the starting
hydrolysis. The recovery of the internal standard gives the
concentration of these amino acids. Time-course hydrolysis
general recovery of the amino acids of the protein solution.
studies are also used with amino acids that are slow to cleave
Free amino acids, ho~ever, do not behave in the same way (e.g., isoleucine and valine). During the hydrolysis time
as protein-bound amino acids during hydrolysis, whose rates course, the analyst will observe a plateau in these residues.
of releas e or destruction are variable. Therefore, the use of an The level of this plateau is taken as the residue
internal standard to correct for losses during hydrolysis may concentration. If the hydrolysis time is too long, the residue
give unreliable results. It will be necessary to take this point concentration of the sample will begin 10 decrease, indicating
into consideration when interpreting the results. Internal
destruction by the hydrolysis conditions.
standards can also be added to the mixture of amino acids
after hydrolysis to correct for differences in sample An acceptable alternative to the time-course study is to
application and changes in reagent stability and fiow rates. subject an amino acid calibration standard to the same
Ideally, an internal standard is an unnaturally occurring hydrolysis conditions as the test sample. The amino acid in
primary amino acid that is commercially available and free form may not completely represent the rate of
inexpensive. It should also be stable during hydrolysis, its destruction of labile amino acids within a peptide or protein
response factor should be linear with concentration, and it during the hydrolysis. This is especially true for peptide
needs to elute with a unique retention time without bonds that are slow to cleave (e.g., De-Val bonds). However,
overlapping other amino acids. Commonly used amino acid this technique will allow the analyst to account for sorne
standards include norleucine, nitrotyrosine, and residue destruction. Microwave acid hydrolysis has been used
ex-aminobutyric acid. and is rapid but requires special equipment as well as special
precautions. The optimal conditions for microwave hydrolysis
Protein Hydrolysis must be investigated for each individual proteinlpeptide
Hydrolysis of protein and peptide samples is necessary for sample. The microwave hydrolysis technique typically
amino acid analysis of these molecules. The glassware used requires only a few minutes, but even a deviation of one
for hydrolysis must be very clean to avoid erroneous results. minute may give inadequate results (e.g., incomplete
Glove powders and fingerprints on hydrolysis tubes may hydrolysis or destruction of labile amino acids) . Complete
cause contamination. To clean glass hydrolysis tubes, boil proteolysis, using a mixture of proteases, has been used but
tubes for 1 h in 1 M hydrochlonc acid or soak tubes in can be complicated, requires the proper control s, and is
concentrated nitric acid or in a mixture of equal volumes of typically more applicable to peptides than proteins.
concentrated hydrochloric acid and nitric acid. Clean
During initial analyses of an unknown protein, experiments Method 4

with various hydrolysis time and temperature conditions are Cysteine/cystine and methionine oxidation is performed with
conducted to determine the optimal conditions . performic acid before the protein hydrolysis .
Method 1 Oxidation solution Use performic acid freshly prepared
Acid hydrolysis using hydrochloric acid containing phenol is by mixing 1 volume of hydrogen peroxide solution
the most common procedure used for proteinlpeptide (30 per cent) and 9 volumes of anhydrous formic acid and
hydrolysis preceding amino acid analysis. The addition of incubating at room temperature for 1 h.
phenol to the reaction prevents the halogenation of tyrosine. Procedure Dissolve the protein/peptide sample in 20 ~lL
Hydrolysis solution 6 M hydrochloric acid containing of anhydrous forrnic acid and heat at 50 oC for 5 minó then
0.1 per cent to 1.0 per cent ofphenoI. add 100 ¡.tL of the oxidation solution. Allow the oxidation to
proceed for 10-30 min. In this reaction, cysteine is converted
Procedure
to cysteic acid and methionine is converted to methionine-
Liquid phase hydrolysis Place the protein or peptide sulfone. Remove the excess reagent from the sample in a
sample in a hydrolysis tube, and dry (the sample is dried so vacuum centrifuge. The oxidised protein can then be acid
that water in the sample will not dilute the acid used for the hydrolysed using Method 1 or Method 2. This technique
hydrolysis). Add 200 IlL of hydrolysis solution per 500 Ilg of may cause modifications to tyrosine residues in the presence
Iyophilised protein. Freeze the sample tube in a dry ice- of halides.
acetone bath, and flame seal in vacuo. Samples are typicaIly
hydrolysed at 110 °C for 24 h in vacuo or in an inert Method 5
armosphere to prevent oxidation. Longer hydrolysis times Cysteine/cystine oxidation is accomplished during the liquid
(e.g., 48 h and 72 h) are investigated if there is a concem phase hydrolysis with sodium azide.
that the protein is not completely hydrolysed. Hydrolysis solution To 6 M hydrochloric acid containing
Vapour phase hydrolysis This is one of the most 0.2 per cent of phenol, add sodium azide to obtain a final
common acid hydrolysis pro ce dures, and it is preferred for concentration of 2 gIL. The added phenol prevents
microanalysis when only smaIl amounts of the sample are halogenation of tyrosine.
available. Contamination of the sample from the acid reagent Liquid phase hydrolysis Conduct the protein/peptide
is also minimised by using vapour phase hydrolysis. Place hydrolysis at about 110 oC for 24 h. During the hydrolysis,
vials containing the dried samples in a vessel that contains an the cysteine/cystine present in the sample is converted to
appropriate amount of hydrolysis solution. The hydrolysis cysteic acid by the sodium azide present in the hydrolysis
solution does not come in contact with the test sample. solution. This technique allows better tyrosine recovery than
Apply an inert armosphere or vacuum (Iess than 200 11m of Method 4, but it is not quantitative for methionine.
mercury or 26.7 Pa) to the headspace of the vessel, and heat Methionine is converted to a mixture of the parent
to about 110 oC for a 24 h hydrolysis time. Acid vapour methionine and its 2 oxidative products, methionine-
hydrolyses the dried sample. Any condensation of the acid in sulfoxide and methionine-sulfone.
the sample vials is to be minimised. After hydrolysis, dry the
Method 6
test sample in vacuo to remove any residual acid.
Cysteine/cystine oxidation is accomplished with dimethyl
Method 2 sulfoxide (DMSO).
Tryptophan oxidation during hydrolysis is decreased by using Hydrolysis solution To 6 M hydrochloric acid containing
mercaptoethanesulfonic acid as the reducing acid. 0.1 per cent to 1.0 per cent of phenol, add dimethyl
Hydrolysis solution 2.5 M mercaptoethanesulfonic acid sulfoxide to obtain a final concentration of 2 per cent V/V.
solution. Vapour phase hydrolysis Conduct the proteinlpeptide
Vapour phase hydrolysis Dry about 1 Ilg to 100 Ilg of hydrolysis at about 110 oC for 24 h. During the hydrolysis,
the proteinlpeptide under test in a hydrolysis tube. Place the the cysteine/cystine present in the sample is converted to
hydrolysis tube in a larger tube with about 200 IlL of the cysteic acid by the DMSO present in the hydrolysis solution.
hydrolysis solution. Seal the larger tube in vacuo (about As an approach to limit variability and compensate for
50 11m of mercury or 6.7 Pa) to vaporise the hydrolysis partial destruction, it is recommended to evaluate the cysteic
solution. Heat the hydrolysis tube to 170-185 oC for about acid recovery from oxidative hydrolysis of standard proteins
12.5 minoAfter hydrolysis, dry the hydrolysis tube in vacuo containing 1-8 mol of cysteine. The response factors from
for 15 min to remove the residual acid. proteinlpeptide hydrolysates are typicaIly about 30 per cent
Method 3 lower than those for non-hydrolysed cysteic acid standards.
Because histidine, methionine, tyrosine, and tryptophan are
Tryptophan oxidation during hydrolysis is prevented by using
thioglycoIlic acid (TGA) as the reducing acid. also modified, a complete compositional analysis is not
obtained with this technique.
Hydrolysis solution 7 M hydrochloric acid containing
1 per cent of phenol, 10 per cent of trifluoroacetic acid and Method 7
20 per cent of thioglycoIlic acid. Cysteine/cystine reduction and alkylation is accomplished by
Vapour phase hydrolysis Dry about 10 ¡.tg to 50 Ilg of a vapour phase pyridylethylation reaction.
the protein/peptide under test in a sample tube. Place the Reducing solution Transfer 83.3 ~lL of pyridine, 16.7 IlL
sample tube in a larger tube with about 200 ~lL of the of 4-vinylpyridine, 16.7 IlL of tributylphosphine, and
hydrolysis solution. Seal the larger tube in vacuo (about 83.3 ¡.tL of water to a suitable container and mix.
50 flill of mercury or 6.7 Pa) to vaporise the TGA. Heat the Procedure Add the proteinlpeptide (between 1 and
sample tube to 166 cC for about 15-30 mino After 100 ¡.tg) to a hydrolysis tube, and place in a larger tube.
hydrolysis, dry the sample tube in vacuo for 5 min to remove Transfer the reducing solution to the large tube, seal in
the residual acid. Recovery of tryptophan by this method vacuo (about 50 ¡.tm of mercury or 6.7 Pa), and heat at
may be dependent on the amount of sample presento about 100 cC for 5 min. Then remove the inner hydrolysis
2014 Appendix III L V-A221
tube, and dry it in a vacuum desiccator for 15 min to Procedure Transfer about 20 ¡.tg of the test sample to a
remove residual reagents. The pyridylethylated sample can hydrolysis tube, and add 5 ¡.tL of the reducing solution.
then be acid hydrolysed using previously described Add 10 IlL of isopropyl alcohol, and then remove all of the
procedures. The pyridylethylation reaction is performed sample liquid by vacuum centrifugation. The sample is then
simultaneously with a protein standard sample containing hydrolysed using Method l. This method has the advantage
1-8 mol of cysteine to evaluate the pyridylethyl-cysteine that other amino acid residues are not derivatised by side
recovery. Longer incubation times for the pyridylethylation reactions, and that the sample does not need to be desalted
reaction can cause modifications to the et.-amino terminal prior to hydrolysis.
group and the 8-amino group of Iysine in the protein. Method 11
Method 8 Asparagine and glutamine are converted to aspartic acid and
Cysteine/cystine reduction and alkylation is accomplished by glutamic acid, respectively, during acid hydrolysis. Asparagine
a liquid phase pyridylethylation reaction. and aspanic acid residues are added and represented by Asx,
Stock solutions Prepare and filter 3 solutions: 1 M Tris- while glutamine and glutamic acid residues are added and
hydrochloride pH 8.5 containing 4 mM disodium edetate represented by Gl:x. Proteins/peptides can be reacted with
(stock solution A), 8 M guanidine hydrochloride (stock bis(l,l-triftuoroacetoxy)iodobenzene (BTI) to convert the
solution B), and 10 per cent of 2-mercaptoethanol (stock asparagine and glutamine residues to diaminopropionic acid
solution C) . and diaminobutyric acid residues, respectively, upon acid
Reducing solution Prepare a mixture of 1 volume of stock hydrolysis. These conversions allow the analyst to determine
solution A and 3 volumes of stock solution B to obtain a the asparagine and glutamine content of a proteinlpeptide in
buffered solution of 6 M guanidine hydrochloride in 0.25 M the presence of aspartic acid and glutamic acid residues.
tris-hydrochloride. Reducing solutions Prepare and filter 3 solutions: a
Procedure Dissolve about 10 Ilg of the test sample in solution of 10 mM triftuoroacetic acid (Solution A), a
50 IlL of the reducing solution, and add about 2.5 IlL of solution of 5 M guanidine hydrochloride and 10 mM
stock solution C. Store under nitrogen or argon for 2 h at triftuoroacetic acid (Solution B), and a freshly prepared
room temperature in the dark. To achieve the solution of dimethylformamide containing 36 mg of BTI per
pyridylethylation reaction, add about 2 IlL of 4-vinylpyridine millilitre (Solution C) .
to the protein solution, and incubate for an additional 2 h at Procedure In a clean hydrolysis tube, transfer about
room temperature in the dark. Desalt the proteinlpeptide by 200 Ilg of the test sample, and add 2 mL of Solution A or
collecting the proteinlpeptide fraction from a reversed-phase Solution B and 2 mL of Solution C. Seal the hydrolysis tube
HPLC separation. The collected sample can be dried in a in vacuo. Heat the sample at 60 DC for 4 h in the dark.
vacuum centrifuge before acid hydrolysis. The sample is then dialysed with water to remove the excess
reagents. Extract the dialysed sample 3 times with equal
Method 9 volumes of butyl aceta te, and then Iyophilise. The protein
Cysteine/cystine reduction and alkylation is accomplished by can then be acid hydrolysed using previously described
a liquid phase carboxymethylation reaction. procedures . The et., p-diaminopropionic and
Stock solutions Prepare as directed for Method 8. et.,y-diaminobutyric acid residues do not typically resolve
Carboxymethylation solution Prepare a 100 gIL solution from the Iysine residues upon ion-exchange chromatography
of iodoacetamide in alcohol. based on amino acid analysis. Therefore, when using ion-
Buffer solution Use the reducing solution, prepared as exchange as the mode of amino acid separation, the
described for Method 8. asparagine and glutamine contents are the quantitative
difference in the aspartic acid and glutamic acid content
Procedure Dissolve the test sample in 50 IlL of the buffer assayed with underivatised and BTI-derivatised acid
solution, and add about 2.5 IlL of stock solution C. Store hydrolysis. The threonine, methionine, cysteine, tyrosine, and
under nitrogen or argon for 2 h at room temperature in the histidine assayed content can be altered by BTI
dark. Add the carboxymethylation solution in a ratio 1.5 fold derivatisation; a hydrolysis without BTI will have to be
per total theoretical content of thiols, and incubate for an performed if the analyst is interested in the composition of
additional 30 min at room temperature in the dark. If the these other amino acid residues of the proteinlpeptide.
thiol content of the protein is unknown, then add 5 IlL of
100 mM iodoacetamide for every 20 nmol of protein Methodologies of Amino Acid Ana1ysis: General
presento The reaction is stopped by adding excess of PrincipIes
2-mercaptoethanol. Desalt the proteinlpeptide by collecting Many amino acid analysis techniques exist, and the choice of
the proteinlpeptide fraction from a reversed-phase HPLC any one technique often depends on the sensitivity required
separation. The collected sample can be dried in a vacuum from the assay. In general, about one-half of the amino acid
centrifuge before acid hydrolysis. analysis techniques employed rely on the separation of the
The S-carboxyamidomethyl-cysteine formed will be free amino acids by ion-exchange chromatography followed
convened to S-carboxyrnethyl-cysteine during acid by post-column derivatisation (e.g., with ninhydrin or
hydrolysis. o-phthalaldehyde) . Post-column derivatisation techniques can
Method 10 be used with samples that contain small amounts of buffer
Cysteine/cystine is reacted with dithiodiglycolic acid or components, (such as salts and urea) and generally require
dithiodipropionic acid to produce a mixed disulfide. between 5 Ilg and 10 Ilg of protein sample per analysis.
The choice of dithiodiglycolic acid or dithiodipropionic acid The remaining amino acid techniques typically involve pre-
depends on the required resolution of the amino acid analysis column derivatisation of the free amino acids (e.g., phenyl
method. isothiocyanate; 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate or o-phthalaldehyde;
Reducing solution A 10 gIL solution of dithiodiglycolic
(dimethylamino) azobenzenesulfonyl chloride;
acid (or dithiodipropionic acid) in 0.2 M sodium hydroxide.
9-ftuorenylmethyl eh loro forma te; and 7-ftuoro-4-nitrobenzo-
2-oxa-l,3-diazole) followed by reversed-phase HPLC. Pre- sodium hypochlorite or chloramine T and post-column
column derivatisation techniques are very sensitive and derivatisation using OPA and a thiol compound such as
usually require between 0.5 ~g and 1.0 ~lg ofprotein sample N-acetyl-L-cysteine or 2-mercaptoethano!. The derivatisation
per analysis but may be infiuenced by buffer salts in the of primary amino acids is not noticeably affected by the
samples . Pre-column derivatisation techniques may also continuous supply of sodium hypochlorite or chloramine T.
result in multiple derivatives of a given amino acid, which Separation of the amino acids on an ion-exchange column is
complicates the result interpretation. Post-colurnn accomplished through a combination of changes in pH and
derivatisation techniques are generally infiuenced less by cation strength. After post-column derivatisation of eluted
performance variation of the assay than pre-column amino acids with OPA, the reactant passes through the
derivatisation techniques. fiuorometric detector. Fluorescence intensity of OPA-
The following methods may be used for quantitative amino derivatised amino acids are monitored with an excitation
acid analysis. Instruments and reagents for these procedures wavelength of 348 nm and an emission wavelength of
are available commercially. Furthermore, many modifications 450 nm.
of these methodologies exist with different reagent The detection limit is considered to be a few tens of
preparations, reaction procedures, chromatographic systems, picomole level for most of the OPA-derivatised amino acids.
etc. Specific parameters may vary according to the exact Response linearity is obtained in the range of a few picomole
equipment and procedure used. Many laboratories will use level to a few tens of nano mole leve!. To obtain good
more than one amino acid analysis technique to exploit the compositional data, samples larger than 500 ng of
advantages offered by each. In each of these methods, the protein/peptide before hydrolysis are recommended.
analogue signal is visualised by means of a data acquisition
system, and the peak areas are integrated for quantification Method 3 - Pre-column PITC Derivatisation
purposes. Phenylisothiocyanate (PITC) reacts with amino acids to form
phenylthiocarbamyl (PTC) derivatives which can be detected
Method 1 - Post-column Ninhydrin Derivatisation with high sensitivity at 254 nm. Therefore, pre-column
Ion-exchange chromatography with post-colurnn ninhydrin derivatisation of amino acids with PITC followed by a
derivatisation is one of the most common methods employed reversed-phase HPLC separation with UV detection is used
for quantitative amino acid analysis. As a rule, a lithium- to analyse the amino acid composition.
based cation-exchange system is employed for the analysis of After the reagent is removed under vacuum, the derivatised
the more complex physiological samples, and the fas ter amino acids can be stored dry and frozen for several weeks
sodium-based cation-exchange system is used for the more with no significant degradation. If the solution for injection is
simplistic amino acid mixtures obtained with protein kept cold, no noticeable loss in chromatographic response
hydrolysates (typically containing 17 amino acid occurs after 3 days.
components). Separation of the amino acids on an ion-
exchange column is accomplished through a combination of Separation of the PTC-amino acids on a reversed-phase
changes in pH and cation strength. A temperature gradient is HPLC with an octadecylsilyl (ODS) column is accomplished
often employed to enhance separation. through a combination of changes in concentrations of
acetonitrile and buffer ionic strength. PTC-amino acids
When the amino acid reacts with ninhydrin, the reactant has eluted from the column are monitored at 254 nm.
a characteristic purple or yellow colour. Amino acids, except
imino acid, give a purple colour, and show an absorption The detection limit is considered to be 1 pmol for most of
maximum at 570 nm. The imino acids such as proline give a the PTC-amino acids. Response Iinearity is obtained in the
yellow colour, and show an absorption maximum at 440 nm. range of 20-500 pmol with correlation coefficients exceeding
The post-colurnn reaction between ninhydrin and amino 0.999 . To obtain good compositional data, samples larger
acids eluted from the column is monitored at 440 nm and than 500 ng of protein/peptide before hydrolysis are
570 nm, and the chromatograrn obtained is used for the recommended.
determination of amino acid composition. Method 4 - Pre-column A QC Derivitisation
The detection limit is considered to be 10 pmol for most of Pre-column derivatisation of amino acids with
the amino acid derivatives, but 50 pmol for the proline 6-arninoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)
derivative. Response linearity is obtained in the range of followed by reversed-phase HPLC separation with
20-500 pmol with correlation coefficients exceeding 0.999. fiuorometric detection is used.
To obtain good composition data, samples larger than 1 ~g AQC reacts with amino acids to form stable, fiuorescent
before hydrolysis are best suited for this amino acid analysis unsymmetric urea derivatives (AQC-amino acids) which are
of proteinlpeptide. readily amenable to analysis by reversed-phase HPLC.
Method 2 - Post-column OPA derivatisation Therefore, pre-column derivatisation of amino acids with
o-Phthalaldehyde (OPA) reacts with primary amines in the AQC followed by reversed-phase HPLC separation with
presence of thiol compound, to form highly fiuorescent fiuorimetric detection is used to analyse the amino acid
isoindole products. This reaction is used for the post-column composition.
derivatisation in analysis of amino acids by ion-exchange Separation of the AQC-amino acids on a reversed-phase
chromatography. The rule of the separation is the same as HPLC with an ODS column is accomplished through a
Method l. combination of changes in concentrations of acetonitrile and
Although OPA does not react with secondary amines (imino buffer ionic strengh. Selective fiuorescence detection of the
acids such as proline) to form fiuorescent substances, the derivatives with an excitation wavelength at 250 nm and an
oxidation with sodium 'hypochlorite or chloramine T allows emission wavelength at 395 nm allows for the direct injection
secondary amines to react with OPA. The procedure employs of the reaction mixture with no significant interference from
a strongly acidic cation-exchange column for separation of the only major fiuorescent reagent by-product,
free amino acids followed by post-column oxidation with 6-aminoquinoline. Excess reagent is rapidly hydrolysed
(tl/2< 15 s) to yield 6-aminoquinoline, N-hydroxysuccinimide
2014 Appendix nI L V-A223
and carbon dioxide, and after 1 min no further derivatisation simultaneous quantification of tryptophan residues by
can take place. previous hydrolysis of the proteinlpeptide with sulfonic acids
Peak areas for AQC-amino acids are essentially unchanged such as mercaptoethanesulfonic acid, p-toluenesulfonic acid
for at least 1 week at room temperature. Therefore AQC- or methanesulfonic acid described in Method 2 under
amino acids have more than sufficient stability to allow for Protein hydrolysis. The other acid-Iabile residues, asparagine
overnight automated chromatographic analysis. and glutamine, can also be analysed by previous conversion
The detection limit is considered to range from about 40 into diaminopropionic acid and diaminobutyric acid,
ftnol to 320 fmol for each amino acid, except for cystein. respectively, by treatment of proteinlpeptide with BTI
The detection limit for cystein is approximately 800 fmo!. described in Method 11 under Protein hydrolysis.
Response linearity is obtained in the range of 2.5-200 ¡lM The non-proteinogenic amino acid norleucine cannot be used
with correlation coefficients exceeding 0.999. Good as an internal standard in this method as this compound is
compositional data can be obtained from the analysis of eluted in a chromatographic regíon crowded with peaks of
derivatised protein hydrolysates derived from as liule as primary amino acids. Nitrotyrosine can be used as an internal
30 ng of proteinlpeptide. standard because it is eluted in a clean region.
Method 5 - Pre-co/umn OPA Derivatisation The detection limit of DABS-amino acid is about 1 pmo!.
As liule as 2-5 pmol of an individual DABS-amino acid can
Pre-column derivatisation of amino acids with
be quantitatively analysed with reliability, and only 10-30 ng
o-phthalaldehyde (OPA) followed by reversed-phase HPLC
of the dabsylated protein hydrolysate is required for each
separation with fluorometric detection is used. This analysis.
technique does not detect amino acids that exist as secondary
amines (e.g., proline) . Method 7 - Pre-co/umn FMOC-C/ Derivatisation
OPA in conjunction with a thiol reagent reacts with primary Pre-column derivatisation of amino acids with
amine groups to form highly fluorescent isoindole products. 9-fluorenyImethyl chloroformate (FMOC-CI) fo llowed by
2-Mercaptoethanol or 3-mercaptopropionic acid can be used reversed-phase HPLC separation with fluorometric detection
as the thio!. OPA itself does not fluoresce and consequentIy is used.
produces no interfering peaks. In addition, its solubility and FMOC-CI reacts with both primary and secondary amino
stability in aqueous solution, along with the rapid kinetics for acids to form highly fluorescent products. The reaction
the reaction, make it amenable to automated derivatisation proceeds under mild conditions in aqueous solution and is
and analysis using an autosampler to mix the sample with the completed in 30 s. The derivatives are stable, only the
reagent. However, lack of reactivity with secondary amino histidine derivative showing any breakdown. Although
acids has been a predominant drawback. This method does FMOC-CI is fluorescent itself, the reagent excess and
not detect amino acids that exist as secondary amines (e.g., fluorescent side-products can be eliminated without loss of
proline). To compensate for this drawback, this technique FMOC-amino acids.
may be combined with another technique described in FMOC-amino acids are separated by a reversed-phase
Method 7 or Method 8. HPLC using an ODS column. The separation is carried out
Pre-column derivatisation of amino acids with OPA is by gradient elution varied linearly from a mixrure of
followed by a reversed-phase HPLC separation. Because of 10 volumes of acetonitrile, 40 volumes of methanol and
the instability ofthe OPA-amino acid derivative, HPLC 50 volumes of acetic acid buffer to a mixrure of 50 volumes
separation and analysis are performed immediately following of acetonitrile and 50 volumes of acetic acid buffer and 20
derivatisation. The liquid chromatograph is equipped with a amino acid derivatives are separated in 20 mino Each
fluorometric detector for the detection of derivatised amino derivative eluted from the column is monitored by a
acids. Fluorescence intensity of OPA-derivatised amino acids fluorometric detector set at an excitation wavelength of
is monitored with an excitation wavelength of 348 nm and an 260 nm and an emission wavelength of 313 nm.
emission wavelength of 450 nm. The detection limit is in the low femtomole range. A linearity
Detection limits as low as 50 fmol via fluorescence have been range ofO .I-50 ¡lM is obtained for most ofthe amino acids.
reported, although the practicallimit of analysis remains at 1 Method 8 - Pre-co/umn NBD-F Derivatisation
pmo!.
Pre-column derivatisation of amino acids with 7-ftuoro-4-
Method 6 - Pre-co/umn DABS-C/ Derivatisation nitrobenzo-2-oxa-l ,3-diazole (NBD-F) followed by reversed-
Pre-column derivatisation of amino acids with phase HPLC separation with ftuorometric detection is used.
(dimethylamino)azobenzenesulfonyl chloride (DABS-CI) NBD-F reacts with both primary and secondary amino acids
followed by reversed-phase HPLC separation with visible to form highly fluorescent products. Amino acids are
light detection is used. derivatised with NBD-F by heating to 60 oC for 5 mino
DABS-CI is a chromophoric reagent employed for the NBD-amino acid derivatives are separated on an ODS
labelling of amino acids. Amino acids labelled with DABS-CI column of a reversed-phase HPLC by employing a gradient
(DABS-amino acids) are highly stable and show an elution system consisting of acetonitrile and aqueous buffer
absorption maximum at 436 nm. mixture, and 17 amino acid derivatives are separated in 35
DABS-amino acids, all naturally occurring amino acid mino e-Aminocaproic acid can be used as an internal
derivatives, can be separated on an ODS column of a standard, because it is eluted in a clean chromatographic
reversed-phase HPLC by employing gradient systems region. Each derivative eluted from the column is monitored
consisting of acetonitrile and aqueous buffer mixrure. by a fluorometric detector set at an excitation wavelength of
Separated DABS-amino acids eluted from the column are 480 nm and an emission wavelength of 530 nm.
detected at 436 nm in the visible regíon. The sensitivity of this method is almost the same as for the
This method can analyse the imino acids such as proline pre-column OPA derivatisation method (Method 5),
together with the amino acids at the same degree of excluding proline to which OPA is not reactive, and might be
sensitivity, DABS-CI derivatisation method permits the advantageous for NBD-F against OPA. The detection limit
for each amino acid is about 10 fmol. Profile analysis can be in which m is the recovered quantity, in nanomoles, of the
achieved with about 1.5 mg of protein hydrolysates in the amino acid under test; M is the total mass, in micrograms, of
pre-column reaction mixture. the protein; and Mrt is the molecular mas s of the unknown
protein.
Data Calculation and Analysis Known protein samples This data analysis technique can
When determining the amino acid content of a be used to investigate the amino acid composition and
proteinlpeptide hydrolysate, it should be noted that the acid protein concentration of a protein sample of known
hydrolysis step destroys tryptophan and cysteine. Serine and molecular mass and amino acid composition using the amino
threonine are partially destroyed by acid hydrolysis, while acid analysis data. When the composition of the protein
isoleucine and valine residues may be only partially cleaved. being analysed is known, one can exploit the fact that sorne
Methionine can undergo oxidation during acid hydrolysis, amino acids are recovered well, while other amino acid
and sorne amino acids (e.g., glycine and serine) are common recoveries may be compromised because of complete or
contaminants. Application of adequate vacuum (Iess than partial destruction (e.g., tryptophan, cysteine, threonine,
200 ¡.¡m of mercury or 26.7 Pa) or introduction of inert gas serine, methionine), incomplete bond cleavage (Le., for
(argon) in the headspace of the reaction vessel during vapour isoleucine and valine) and free amino acid contamination
phase hydrolysis can reduce the level of oxidative destruction. (i.e., by glycine and serine).
Therefore, the quantitative results obtained for cysteine, Because those amino acids that are recovered best represent
tryptophan, threonine, isoleucine, valine, methionine, glycine, the protein, these amino acids are chosen to quantify the
and serine from a protein/peptide hydrolysate may be variable amount of protein. Well-recovered amino acids are, typically,
and may warrant further investigation and consideration. aspartate-asparagine, glutamate-glutamine, alanine, leucine,
Amino Acid Mole Percent This is the number of specific phenylalanine, Iysine, and arginine. This list can be modified
amino acid residues per 100 residues in a protein. This result based on experience with one's own analysis system. Divide
may be useful for evaluating amino acid analysis data when the quantity, in nanomoles, of each of the well-recovered
the molecular mas s of the protein under investigation is amino acids by the expected number of residues for that
unknown. This information can be used to corroborate the amino acid to obtain the protein content based on each well-
identity of a protein/peptide and has other applications. recovered arnino acid. Average the protein content results
Carefully identify and integrate the peaks obtained as calculated. The protein content determined for each of the
directed for each procedure. Calculate the mole percent for well-recovered amino acids should be even1y distributed
each amino acid present in the test sample using the about the mean. Discard protein content values for those
formula: amino acids that have an unacceptable deviation from the
mean. Typically greater than 5 per cent variation from the
100ru mean is considered unacceptable. Recalculate the mean
r protein content from the remaining values to obtain the
protein content of the sample. Divide the content of each
in which r u is the peak response, in nanomoles, of the arnino amino acid by the calculated mean protein content to
acid under test; and r is the sum of peak responses, in determine the amino acid composition of the sample by
nanomoles, for all amino acids present in the test sample. analysis.
Comparison of the mole percent of the amino acids under Calculate the relative compositional error, in percentage,
test to data from known proteins can help establish or using the formula:
corroborate the identity of the sample protein.
100m
Unknown Protein Samples This data analysis technique
can be used to estimate the protein concentration of an ms
unknown protein sample using the amino acid analysis data.
in which m is the experimentally determined quantity, in
Calculate the mass, in micrograms, of each recovered amino
acid using the formula: nanomoles per amino acid residue, of the amino acid under
test; and ms is the known residue value for that amino acid.
The average relative compositional error is the average of the
mMr
absolute values of the relative compositional errors of the
1000 individual amino acids, typically excluding tryptophan and
cysteine from this calculation. The average relative
in which m is the recovered quantity, in nano moles, of the compositional error can provide important information on
amino acid under test; and M r is the average molecular mass the stability of analysis run over time. The agreement in the
for that amino acid, corrected for the mas s of the water amino acid composition between the protein sample and the
molecule that was eliminated during peptide bond formation. known composition can be used to corroborate the identity
The sum of the masses of the recovered amino acids will give and purity of the protein in the sample.
an estimate of the total mass of the protein analysed after
appropriate correction for partially and completely destroyed
amino acids. 1f the molecular mass of the unknown protein is
available (i.e., by SDS-PAGE analysis or mas s spectroscopy),
the amino acid composition of the unknown protein can be
predicted. Calculate the number of residues of each amino
acid using the formula:
m
2014 Appendix III M V-A225
This heterogeneity in glycosylation results in a set of

M. Glycan Analysis of Glycoproteins glycoforms for one specific glycoprotein. These variations
(Ph . Eur. method 2.2.59) arise because, unlike transcription and translation,
glycosylation is a non-template post-translational modificatÍon
1 Introduction process. The glycosylation partem at a given site depends on
Glycan analysis is a test to analyse glycan moieties of many factors including the cell-specific and/or growth-
glycoproteins. It may involve: dependent availability of glycosyltransferases and exo-
~ whole glycoprotein analysis; glycosidases found in the Golgi apparatus and endoplasmic
reticulum. Protein glycosylation is also influenced by the
~ separation and detection of protein glycoforms;
protein structure, the production process, the host-vector
- analysis of glycopeptides obtained after enzymatic expression system and the cell culture conditions.
treatment of the glycoprotein;
- analysis of released glycans obtained after chemical or 2 Glycan analysis procedures
enzymatic treatment of the glycoprotein. Heterogeneity in glycosylation can be assessed by 4 distinct
Monosaccharide analysis may complement information and complementary approaches:
obtained by glycan analysis. - analysis of the intact glycoprotein;
Glycosylation can playa predominant role in determining the - analysis of glycopeptides;
function, pharmacokinetics, pharmacodynamics, stability, and
- analysis of released glycans;
immunogenicity of biotherapeutics. Glycosylation, unlike
transcription, is a non-template-driven enzymatic - monosaccharide analysis.
modification process that results in glycan heterogeneity. The present section provides methods and general
The manufacturing procedure also has an influence on requirements used for glycan analysis of glycoproteins
glycan heterogeneity. Glycoprotein glycan analysis may containing N- and O-linked glycans.
therefore be an important test to identify variations in the Glycan analysis is usually a multistep process. There are
glycosylation partem of the glycoprotein and/or monitor the numerous methodologies for glycan analysis. This variety is a
consistency of the glycosylation partem during production. consequence of the diversity and complexity of glycan
Glycan analysis can be a comparative procedure, because the structures, of the available technologies and detection
information obtained, compared to a similarly treated systems, and of the wide range of approaches depending on
reference substance, confirms product consistency. the level of information required.
This chapter provides approaches used for glycoprotein Figure 2.2.59.-1 provides an overview of glycan analysis
glycan analysis and requirements for the application of analytical procedures that can be employed to apply the
methods and validation of methods. chosen approach( es) . Many variations of the same techniques
Glycan analysis is not a single general method, but involves and conditions are available depending on the glycan
the application of specific procedures and the development of structures and origino
specific glycan maps for each unique glycoprotein. Specific Iso1ation and purification Isolation and purification may
procedures are therefore indicated in relevant specific be necessary for analysis of bulk drug substances or dosage
monographs. forms containing interfering excipients, and, when required,
will be described in the specific monograph.
1-1 Protein glycosylation
There are 3 main types of enzymatic glycosylation found in 2-1 Analysis ofintact glycoprotein
proteins: Analysis of the intact glycoprotein provides information on
- N-glycosylation, which involves the addition of the overal! partem of glycosylation of the glycoprotein.
oligosaccharides to the nitrogen on the terminal amide This approach provides limited information when the
group of asparagine; molecule is large and contains multiple glycosylation sites.
- O-glycosylation, which involves the addition of Methods such as capillary electrophoresis (CE) (2.2.47) and
oligosaccharides to the hydroxyl groups of serine, mas s spectrometry (MS) (2.2.43) can be used. Size-based
threonine, and/or hydroxyproline; techniques, such as size-exclusion chromatography (2.2.30)
- C-glycosylation, which involves the addition of an and sodium dodecyl sulfate polyacrylamide gel
ex-mannopyranose to the C2-carbon of the indole ring of electrophoresis (SDS-PAGE) (2.2.31), may provide
tryptophan. information on the glycosylation status of a protein. If the
degree of sialylation significantly contributes to the biological
Non-enzymatic additions, also known as glycation, can occur
activity of the glycoprotein, ion-exchange chromatography
when proteins are incubated with reducing sugars.
(2.2.46), isoelectric focusing (IEF) (2.2.54) or CE (2.2.47)
This chapter describes analytical methods for the N- and may be performed to monitor sialylation. The technique
O-linked glycosylations, which are the most commonly found must be chosen according to its suitability to provide a
in glycoprotein medicinal products. reliable correlation between the degree of sialylation and the
1-2 Heterogeneity of the protein glycosylation bioactivity of the producto
Different levels of glycan heterogeneity can appear during the 2-2 Analysis of glycopeptides
production of glycoproteins. This heterogeneity may result Analysis of glycopeptides provides information on site-specific
from variations: glycosylation properties, on the degree of occupancy, and on
- in the degree of occupancy (ful!, partial, unoccupied); the oligosaccharide structures. It involves proteolytic
- in the type of glycosylation (N- or O-linked); digestion of the glycoprotein. Approaches to site-specific
- in the oligosaccharide structures (extensions, branching cleavage of the protein backbone are given in general chapter
and linkage). 2.2.55. Peptide mapping.
V-A226 Appendix III M 2014
ANAL YSIS OF INTACT GLYCOPROTEIN
MS
IEF
CE
--+ lon-exchange chromatography
Size-excluslon chromatography
SDS-PAGE
ANALYSIS OF GLYCOPEPTIDES
Direcl analysis
I GLYCOPROTEIN I
JSeparalion prior lo direct analysisl MS
Proleolysis
LC/CE I
PRETREATMENT
I
.. i
(isolalion & purificalion) --+ Deglycosylation
-
if necessary , I of Ihe digesl
.
ANAL YSIS OF RELEASED GLYCANS Analysis of unlabelled glycans
HPAEC-PADI
DeSiaIYlali0rt-1 PG~SMS
Release of glycans
r---+ 0- and/or N-línked glycans:
enzymes or chemicals n
Analysis of labelled glycans
Exo-glycosidases
treatment
I
Labelling
of glycans ~ Separalioni ~~
l LC/CE r CE
MS
LC
ANALYSIS OF MONOSACCHARIDES
HPAEC-PAD
--- Acid
hydrolysis H Fluorophore
labelling
PGC-MS
LC
CE
--+
HColorimetric melhodsj
Legend: LC: Liquid chromatography

CE: Capillary electrophoresis MS : Mass spectrometry
HPAEC-PAD : High-pH anion-exchange chromatography with pulsed PGC: Porous graphite chromatography
amperometric detection SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
IEF: Isoelectric focusing
Figure 2.2.59.-1. - Overview of glycan analysis procedures
After proteolysis of the glycoprotein, the foIlowing sequentiaIly to direct analysis. Separation techniques such as
approaches can be chosen. liquid chromatography (LC) (2.2.29) and CE (2.2.47) are
Direct analysis by MS (2.2.43) Care should be taken suitable. These techniques may be interfaced with MS to
that the glycopeptide signal is not suppressed due to the aIlow online MS measurements.
presence of other peptides, where glycopeptides represent a Deglycosylation oi the glycopeptides Identification of
minor portion of the total peptide mixture and where signal the different glycosylation sites of a glycoprotein is made
intensities are lower than those of non-glycosylated peptides. possible by comparing peptide maps obtained by proteolytic
Separation prior to analysis by MS This additional step digestion of the intact glycoprotein to those obtained when
overcomes the problems raised aboye. Enrichment or the glycoprotein is deglycosylated previously or folIowing
fractionation techniques can be used either in paraIlel with or proteolytic digestion. The peptide mass gives information
2014 Appendix III M V-A227
about the glycosylation sites and by calculating the mas s relating the peak area of each glycan to the total peak area of
difference between the intact glycopeptide and the all glycans in the map.
deglycosylated glycopeptide, it is possible to obtain
information about the attached glycans conceming Table 2.2.59 -1. - Examples of enzymatic cleavage agents
composition and heterogeneity. Approaches to Agents Specificity
deglycosylation of the protein backbone are given in section
N-Iinked glycans release
2-3-1. A separation step can be performed after or before
deglycosylation. Hydrolysis of an N'-(acetyl-~D-
glucosaminyI)asparagIne residue
2-3 Analysis of released glycans in which the glucosamine residue
Peptlde-N'-(N-acetyl~gIucosaminyl)- may be further glycosylated,
Analysis of relea sed glycans provides a convenient way to asparagIne amidase (EC 3.5.1.52) lo yield a (substituted)
obtain information on the various populations of glycans N-acetyl~D-glucosaminylamine and
a peptlde containing an aspartate
present on the protein (bi-, tri-, and tetra-antennary profile) . residue
The degree of sialylation can also be addressed at this stage. Release of N-glycan chain bul no
Depending on the chosen method, prior - Peptide N-glycosidase F (PNGase F) release of N-glycan chain containing
(al-3)-linked core fucose
derivatisation/labelling may be needed to allow the detection
of the glycans. Release of N-glycan chain containing
- Peplide N-glycosidase A (PNGase A)
(al-3)-linked core fucose
Analysis of released glycans gene rally involves the release and Endohydrolysis oi the
purification of glycans from the reaction mixture, followed by lIIannosyl-glycoprotein
N,N'--diacetylchitobiosyl unil in
the labelling/derivatisation of the glycans, where needed; high-mannose
endo-~N-acetylgIucosaminidase
glycopeptldes/glycoproteins
the glycans are then profiled (fractionation or separation) . (EC 3.2.1.96)
containing the
-[lIIan(GlcNAc2JAsn structure
2-3-1 RELEASE OF GLYCANS
- Endo-~N-acetylglucosaminidase F Release of high-mannose, hybrid and
The selection of the approach used for the release of glycans (endo F) complex oligosaccharides
will depend on the glycoprotein under test. The cleavage . Endo-~N-acetylglucosaminidase H Release of high·mannose, hybrid
agent to be employed is chosen according to the type of (endo H) oligosaccharides
cleavage needed and level of information required. Enzymatic O-Iinked gIycans release
or chemical cleavage may be used. Table 2.2.59.-1 gives a
Glycopeptlde Hydrolysis oi lerminal D-galactosyl-
non-exhaustive list of enzymatic cleavage agents and their a-N-acetylgalactosaminidase (EC N-acetyl-a-D-galactosaminidic
specificity. 3.2.1.97)' residues
Digestion efficiency is generally dependent on the , This enzyme has limited usage because of its high substrate specificity.
accessibility of the glycans on the protein and hence the
protein can be denatured to maximise glycosylation site PGC can also be used to separate native glycans because of
exposure, unless it is desirable to distinguish between surface its higher selectivity compared to the conventional non-polar
and buried glycans . columns. A PGC-electrospray-ionisation-MS approach can
Che mi cal cleavage agents might also be used, using for be applied for direct glycan analysis.
example hydrazine or alkaline borohydride for ~-elimination. 2-3-2-2 ANALYSIS OF LABELLED GLYCANS
2-3-2 ANALYSIS OF GLYCANS Labelling 01 glycans
Released glycans can be analysed or profiled by The type of derivatisation carried out will depend on the
chromatographic, electrophoretic and mass spectrometric method used to detect glycans: UV or fiuorescent.
techniques, and in general by a combination of these Derivatisation with fiuorescent labels is the most commonly
techniques. The choice of the method can be grouped used technique for labelling glycans at their reducing end by
according to the nature of the glycans and level of reductive amination. One label can be attached to every
information required. single mono- and oligo-saccharide, allowing the
Analysis of glycans provides information on the various determination of molar quantities. Table 2.2.59.-2 gives a
populations of glycans present on the protein (high-mannose, non-exhaustive list of commonIy used fiuorescent labels and
hybrid, complex) . Information on the relative amounts of suitable analytical techniques.
branched structures might be obtained by analysis of
desialylated glycans. Table 2.2.59.-2. - Examples of fluorescent labels and suitable
A separation step may be required. It implies the use of LC techn iques
(2.2.29) and CE (2.2. 47) as intermediate techniques. Name Acronym Analytical techniques
LC (2.2.29) can be used preparatively with individual 2-Aminobenzoic acid 2-AA LC (2.2.29), MS (2.2.43)
fractions being collected (usually labelling is required) or can
2-Aminobenzamide 2-AB LC (2.2.29), MS (2.2.43)
be directly coupled to MS (2.2.43).
2-3-2-1 ANALYSIS OF U NLABELLED GLYCANS 2-Aminopyridine 2·AP LC (2.2.29), lIIS (2.2.43)
Native glycans can be analysed by high-pH anion-exchange 2-Amino-9(lOH)-acridinone A!IIAC Gel electrophoresis (2.2.23)
chromatography with pulsed amperometric detection Trisodium 8-aminopyrene-l,
(HPAEC-PAD) , porous graphite chromatography (PGC) APTS CE (2.2.47)
3,6-trisulfonic acid
and MS (2.2.43).
HPAEC-PAD has high sensitivity and can also separate sorne Permethylation of glycans may also be used when MS
linkage isomers. Response factors of the different signals are (2.2.43) is used alone for detection. It is based on the
not equal for the different oligosaccharide structures. methylation of the oligosaccharides.
Absolute quantification of the glycan is not possible unless an
oligosaccharide reference library is available. Quantification
can be obtained by comparison with a well-characterised
reference standard of the substance being tested, or by
V-A228 Appendix III M 2014
A nalysis of labelled glycans 3- 1-1 ABSOLUTE CONFIRMATION OF lDENTITY

Labelled glycans can be analysed by analytical techniques Absolute confirmation of the identity of glycan structures is
such as LC (2.2.29), CE (2.2.47) and MS (2.2.43). typically achieved during product development, and should
not necessarily be the target of routine analysis. Identity of
According ro the separation properties of the glycans, glycans
the analytical target will be assigned by reference to a known
can be profiled and quantified by several LC (2.2.29)
molecular property of the molecule. Such absolute
systems using an appropriate labe!: reversed-phase
identification of individual structures can require multi-step
(separation by hydrophobicity), normal-phase (separation by
approaches using enzymatic and chemical reactions,
size), and anion-exchange (separation by charge) Le.
separation techniques and online or offiine detection
2-4 Monosaccharide analysis methods, and will most commonly use the charge-to-mass
Monosaccharide analysis provides information on the ratio of a molecular ion, determined using a suitable mass
monosaccharide composition of a glycoprotein. Analysis of spectrometric method as the final basis for structure
monosaccharides can be performed using either colorirnetric assignment.
or separation methods. 3-1-2 COMPARATIVE CONFIRMATION OF lDENTITY
2-4-1 COLORIMETRIC METHODS During routine application of the analytical method, the
The colorimetric methods, which are based on chemical identity of the analytical target may be confirmed by
staining, provide information on the quantity of specific comparison with process or system suitability reference
classes of sugars such as sialic acids, neutral sugars and standards. These may be generated from known, well-
hexosamines. characterised glycoproteins, which may be of the same
2-4-2 SEPARATION METHODS general class as the product being tested (e.g. fetuin for
The separation methods generate quantitative information on complex N-linked glycoproteins), or may be derived from a
the overall monosaccharide composition. The methods well-characterised batch of the product being tested, which
require acid hydrolysis pre-treatrnent of the oligosaccharide has been established as a reference standard. The following
chains of the intact glycoprotein or released glycans, prior to considerations apply ro comparative assignment of structural
analysis. To releas e sialic acids, mild acid hydrolysis or identity:
enzymatic treatment is employed. The hydrolysis step is a - in the case of a validated high reproducibility of the
significant source of variability and may require retention -times, the absolute retention times can be used
product-specific validation. for correct assignment;
Methods for separation and quantification of - altematively, a glycan marker can be injected at the
monosaccharides include: beginning and at the end of the testing sequence and
- the use ofHPAEC-PAD and PGC-MS, which allow the checked for any drifts in the retention times; based on
determination of molar quantities of native these reference chromarograms the glycans of the test
monosaccharides (sialic acids, neutral sugars and alcohol samples can be assigned;
sugars); - in cases where no standard is available to assign all glycan
- ftuorophore labelling of monosaccharides followed by peaks in the test sample, absolute or normalised retention
separation methods such as reversed-phase or times can be used to monitor and label unidentified
ion-exchange chromatography, or CE. glycan peaks.
3-2 Confirmation oi compliance oi the substance being
3 Evaluation and analysis of data tested with qualitative requirements
Data obtained from analytical methods for the analysis of At this level of evaluation, the analytical results obtained with
glycans can be analysed and evaluated for 3 different the product being tested are evaluated to demonstrate
purposes: compliance with spec:ifications. Typically this is achieved by
- confirmation of identity of individual structures or families comparison with data obtained in parallel using a reference
of structures; standard of the substance being tested, In evaluating the data
- confirmation of compliance of the substance being tested it is necessary:
with qualitative requirements; - to establish that the analytical result obtained using the
- confirmation of compliance of the substance being tested reference standard is broadly comparable ro the expected
with quantitative requirements. result, ro verify the suitability of the system; for example,
in a glycan mapping pro ce dure, this would be achieved by
Specific considerations with respect to refetence standards
comparison of the map obtained with the reference
and method development of each level of analysis are set out
substance with a provided specimen map obtained during
in sections 4 and 5 respectively.
establishment of the reference substance, and by ensuring
3-1 Confirmation oi identity oi individual structures or compliance with all stated system suitability criteria;
iamilies oi structures - ro demonstrate sirnilarity of the maps obtained with the
The analytical target for a glycan analysis method may be an reference substance and the test substance, using any
individual monosaccharide (e.g. sialic acid, fucose), a defined specific compliance criteria given in the specific
oligosaccharide structure (e.g. tetra-sialylated, tetra-antennary monograph.
glycan) or a family of structures sharing a common analytical
3-3 Confirmation oi compliance oi the substance being
feature (e.g. tetra-sialylated glycans, tri-antennary glycans,
tested with quantitative requirements
glycoprotein isoforms with the same charge). Confirmation of
3-3-1 QUANTITATIVE MEASUREMENT OF ANALYTE LEVELS
the identity of the analytical target is an essential step in the
AND EXPRESSIO N OF RESULTS
analysis and evaluation of data, and can be achieved
absolutely, by verification of molecular structure, or In sorne cases, e.g. measurement of sialic acid or other
comparatively, by comparison with an appropriate reference monosaccharides, data can be expressed in order to obtain a
standard. molar ratio of sialic acid to glycoprotein. Data is calculated
2014 Appendix In M V-A229
Glycan analysis necessary for the glycoprotein
Design glycan analysis methods
Small protein, limited glycosylation >---l~ Analysis of intact glycoprotein (e.g.

and/or simple structures? mass spectrometric analysis)
Ves L-----------r-----------~
No
Large molecule, complex branched glycans and/or

multiple glycosylation sites
Knowledge of site-specific Ves

glycosylation required?
No
Negative charge content determination No Detailed glycan

Sialic acid and/or monosaccharides analysis required?
determination
Ves
Design methods for analysis of cleaved glycans (e.g. antennary

profile, identification of individual structure)
Figure 2.2.59.-2. - Guidance on mefhods fo be used when glycan analysis is required
by reference to a reference standard for sialic acid and to a The reference standard used for compliance of the
validated method of protein determination. Either the glycoprotein under test is a preparation of the substances
internal or external standard method may be used (see being tested. Ir is noted that glycan analysis procedures
general chapter 2.2.46. Chromatographic separation techniques). described in specific monographs prescribe the use of a
3-3-2 QUANTITATIVE EXPRESSIONS OF SEPARATION reference standard for the substance being tested and for
PRO FILE which the glycan analysis procedure has been validated.
Profiles or distribution patterns may be expressed numerically
in a number of ways, induding the normalisation procedure; 5 Points to consider in method development
the percentage content of each analytical target, e.g. glycan This section provides rneans for measuring the overall
entity, is calculated by determining the response of the glycan performance of the method during development. The extent
entity as a percentage of the total response of all the entities, of method development and analytical validation is selected
exduding those due to solvents or any added reagents, and on the basis of their suitability for a specific product.
those below the disregard limito In addition, numerical Depending on the chosen approach, several steps are
expressions such as the Z number, which are method- and necessary for glycan analysis, for example:
product-specific and defined in specific monographs, can be - isolation and purification (or desalting) of the
used. glycoprotein;
4 Reference standards - enzymatic (or chemical) treatment of the glycoprotein to
selectively release either N- or O-linked glycans from the
Reference standards for glycan analysis serve 2 functions: the protein backbone;
verification of the suitability of the system and the
- isolation and purification of the released glycans;
confirmation that the artide under test complies with
specified requirements. - verification of released sialic acid and monosaccharide
The reference standards used for system suitability may be: residues;
- chromophore labelling of the released glycans;
- a reference substance for the substance being tested;
- glycan moities liberated from a fully characterised - separation of the glycans, native or ftuorescence labelled;
reference standard of the substance being tested; - glycan identification and quantification (e.g. determination
- well-characterised glycan moities liberated from of the Z number);
glycoproteins (e.g. fetuin, IgG); - determination of site occupancy based on relative
quantities of glycosylated and non-glycosylated peptides.
- glycan markers characterised for identity and purity.
V-A230 Appendix In M 2014
Protein isolation and purification Isolation and ensure the quality of the glycoprotein and is set up during
purification of the glycoprotein from its matrix may be the development phase of the product.
necessary to remove all interfering substances Figure 2.2.59.-2 provides guidance in the choice of methods
(e.g. excipients, salts) and, when required, will be specified to be used when glycan analysis is required.
in the specific monograph. This must be performed in a
reproducible manner in order ro guarantee a quantitative
recovery of the protein.
Release and isolation of oligosaccharides The approach
chosen for the release of glycans will depend on the protein
under test and will be based on the types of glycosylation,
i.e. N- or O-linked glycosylation. Non-compendial
approaches available for the release of glycans must be
optimised in order to ascertain a quantitative profiling of all
glycan entities. Facrors that impact c1eavage efficiency, such
as enzyme-to-protein concentration ratio, temperature,
reaction time course, and denaturation of protein prior to
digestion, must be optimised.
Ir is noteworthy that the enzymatidchemical reaction must
not alter the glycan composition, e.g. not destroy sialic acid
residues. Where there is more than one glycosylation site, the
enzymatic treatment should proportionally releas e all
oligosaccharide moieties attached to the protein, independent
of their structure and their individual position in the protein.
Reproducible recovery of all glycan entities from the reaction
mixture must be confirmed.
Derivatisation of released glycans Derivatisation is
usually carried out according ro non-compendial prorocols.
Therefore, the reproducible derivatisation of all glycan
entities must be verified. This may be achieved through
optimisation of the reaction conditions such as amount of
the derivatisation reagent, reaction temperature and time.
The derivatisation reaction must not change the glycan
composition, e.g. not destroy sialic acid residues.
Separation, identification and system suitability The
methods employed for glycan analysis must be capable of
detecting and separating different glycan moieties ro ascertain
a reliable identification and quantification.
The acceptance criteria for system suitability, which also
cover glycan c1eavage, recovery and analysis, depend on the
critical test parameters that affect the outcome of the resulto
A comparison berween the glycan map of the substance
under test and that of a reference substance, being treated in
the same conditions, is an indicaror ro evaluate the
performance of the analytical procedure. In order to further
confirm the obtained results, the analyses may be repeated
with an orthogonal method. The use of a reference standard
(e.g. reference substance of the product being examined,
system suitability glycan marker) is essential in the
establishment of system suitability parameters and validation
of the analytical procedure.
Reproducibility of quantitative expression (e.g. Z number
estimation) of glycan profiles must be verified.
Determination of site occupancy based on relative
quantities of glycosylated and non-glycosylated
peptides Where site occupancy is estimated by comparison
of glycosylated and non-glycosylated peptides from an
enzymatically dígested glycoprotein, reproducible c1eavage of
both forms of the peptide must be demonstrated.
6 Glycan analysis decision-making framework

This decision-making framework is given for information and
does not constitute a mandarory part of the European
Pharmacopoeia. .
The choice of procedures used to analyse glycans is
established according ro the level of information required to
2014 Appendix IV A V-A231
of different particle sizes and shapes that are found in the

Appendix IV real samples. Due to formazin's reproducibility, scattering
characteristics and traceability, instrument calibration
algorithms and performance criteria are mostly based on this
A. Clarity 01 Solution standard.
Instrumental methods
VISUAL METHOD
Using identical test-tubes of colourless, transparent, neutral Introduction
glass with a fiat base and an internal di ame ter of 15-25 mm, The degree of opalescence may also be determined by
compare the liquid to be examined with a refe:¡;ence instrumental measurement of the light absorbed or scattered
suspension freshly prepared as described below, the depth of on account of submicroscopic optical density inhomogeneities
the layer being 40 mm. Compare the solutions in diffused of opalescent solutions and suspensions. 2 such techniques
daylight 5 min after preparation of the reference suspension, are nephelometry and turbidimetry. For turbidity
viewing vertically against a black background. The diffusion measurement of coloured samples, ratio turbidimetry and
of light must be such that reference suspension I can readily nephelometry with ratio selection are used.
be distinguished from water R, and that reference suspension The light scattering effect of suspended particles can be
II can readily be distinguished from reference suspension 1. measured by observation of either the transmitted light
A liquid is considered clear if its clarity is the same as that of (turbidimetry) or the scattered light (nephelometry). Ratio
water R or of the solvent used when examined under the turbidimetry combines the principIes of both nephelometry
conditions described aboye, or if its opalescence is not more and turbidimetry. Turbidimetry and nephelometry are useful
pronounced than that of reference suspension 1. for the measurement of slightly opalescent suspensions.
Hydrazine sulfate solution Dissolve l.0 g of hydrazine Reference suspensions produced under well-defined
conditions must be used. For quantitative measurements, the
sulfqte R in water R and dilute to 100.0 mL with the same
construction of calibration curves is essential, since the
solvent. Allow to stand for 4-6 h.
relationship between the optical properties of the suspension
Hexamethylenetetramine solution In a 100 mL aqd the concentration of the dispersed phase is at best semi-
ground-glass-stoppered fiask, dissolve 2.5 g of
empirica!.
hexamethylenetetramine R in 25.0 mL of water R.
The determination of opalescence of coloured liquids is done
Primary opalescent suspension (formazin suspension) with ratio turbidimeters or nephelómeters with ratio
To the hexamethylenetetramine solution in the fiask add selection, since colour provides a negative interference,
25.0 mL of the hydrazine sulfate solution. Mix and allow to attenuating both incident and scattered light and lowering the
stand for 24 h. This suspension is stable for 2 months, turbidity value. The effect is so great for even moderately
provided it is stored in a glass container free from surface coloured samples that conventional nephelometers cannot be
defects. The suspension must not adhere to the glass and
used.
must be well mixed before use.
The instrumental assessment of clarity and opalescence
Standard of opalescence Dilute 15.0 mL of the primary provides a more discriminatory test that does not depend on
opalescent suspension to 1000.0 mL with water R. This the visual acuity of the analyst. Numerical r'esults are more
suspension is freshly prepared and may be stored for up ro useful for quality monitoring and process control, especially
24 h. in stability studies. For example, previous numerical data on
Reference suspensions Prepare the reference suspensions stability can be projected to detern1ine whether a given batch
according to Table 2.2.l.-l. Mix and shake before use. of dosage formulation or active pharmaceutical ingredient will
exceed shelf-life limits prior to the expiry date.
Table 2.2.1.-1
Nephelometry
II III IV
When a suspension is viewed at right angles to the direction
Standard of opalescence 5.0 mL 10.0 mL 30.0 mL 50.0 mL of the incident light, the system appears opalescent due to
Water R 95.0 mL 90.0 mL 70.0 mL 50.0 mL the refiection of light from the particles of the suspension
(Tyndall effect) . A certain portion of the light beam entering
a turbid liquid is transmitted, another portion is absorbed
Turbidity standard The formazin suspension prepared by and the remaining portion is scattered by the suspended
mixing equal volumes of the hydrazine sulfate solution and particles. If measurement is made at 90° ro the light beam,
the hexamethylenetetramine solution is defined as a 4000 the light scattered by the suspended particles can be used for
NTU (nephelometric turbidity units) primary reference the determination of their concentration, provided the
standard. Reference suspensions 1, II, III and IV have values number and size of particles infiuencing the scattering remain
of3 NTU, 6 NTU, 18 NTU and 30 NTU respectively. constant. The reference suspension must maintain a constant
Stabilised formazin suspensions that can be used to prepare degree of turbidity and the sample and reference suspensions
stable, diluted turbidity standards are available commercially must be prepared under identical conditions. The Tyndall
and rriay be used after comparison with the standards effect depends upon both the number of particles and their
prepared as described. size. Nephelometric measurements are more reliable in low
Formazin has several desirable characteristics that make it an turbidity ranges, where there is a linear relationship between
excellent turbidity standard. It can be reproducibly prepared nephelometric turbidity unit (NTU) values and relative
from assayed raw materials. The physical characteristics make detector signals. As the degree of turbidity increases, not all
it a desirable light-scatter calibration standard. The formazin the particles are exposed to the incident light and the
polymer consists of chains of different lengths, which fold scattered radiation of other particles is hindered on its way to
into random configurations. This results in a wide assay of the detector. The maximum nephelometric values at which
particle shapes and sizes, which analytically fits the possibility reliable measurements can be made lie in the range of
V-A232 Appendix IV A 2014
1750-2000 NTU. Lineariry must be demonstrated by filament colour temperature of 2700 K, or IR LED having
constructing a calibration curve using at least 4 an emission maximum at 860 nm with a 60 nm spectral
concentrations. bandwidth. Other suitable light sources may also be used.
Silicon photodiodes and photomultipliers are commonly used
Turbidimetry
as detectors and record changes in light scattered or
The optical property expressed as turbidity is the interaction transmitted by the sample. The light scattered at 90 ± 2.5°
between light and suspended particIes in liquid. This is an is detected by the primary detector. Other detectors are
expression of the optical property that causes light to be those to detect back and forward scatter as well as
scattered and absorbed rather than transmitted in a straight transmitted light. The instruments used are calibrated against
line through the sample. The quantity of solid material in standards of known turbidity and are capable of automatic
suspension can be determined by the measurement of the determination of turbidity. The test results expressed in
transmitted light. A linear reIationship between turbidity and NTU units are obtained directIy from the instrument and
concentration is obtained when the particIe sizes are uniform compared to the specifications in the individual monographs.
and homogeneous in the suspension. This is true only in very
Instruments complying with the following specifications are
dilute suspensions containing small particIes. Linearity
suitable.
between turbidity and concentration must be established by
constructing a calibration curve using at least 4 - M easuring units: NTU. NTU is based on the turbidity of a
concentrations. primary reference standard of formazin. FTU (Formazin
Turbidity Units) or FNU (Formazin Nephelometry Units)
Ratio Turbidimetry are also used, and are equivalent to NTU in low regions
In ratio turbidimetry the relationship of the transmission (up to 40 NTU). These units are used in a1l3
measurement to the 90° scattered light measurement is instrumental m ethods (nephelometry, turbidimetry and
determined. This procedure compensates for the light that is ratio turbidimetry).
diminished by the colour of the sample. The infiuence of the - Measuring range: 0.01-1100 NTU.
colour of the sample may also be eliminated by using an
infrared light-emitting diode (IR LED) at 860 nm as the light
- Resolution: 0.01 NTU within the range of 0-10 NTU, 0.1
NTU within the range of 10-100 NTU, and 1 NTU for
source of the instrumento The instrument's photodiode
the range > 100 NTU. The instrumentis calibrated and
detectors receive and measure scattered light at a 90° angle
controIled with reference standards of formazin.
from the sample as weIl as measuring the forward scatter
(light refiected) in front of the sample along with the - Accuracy: 0-10 NTU: ± (2 per cent of reading + 0.01)
measurement of light transmitted directIy through the NTU. 10-1000 NTU: ± 5 per cent.
sample. The measuring results are given in NTU(ratio) and - Repeatability: 0-10 NTU: ± 0.01 NTU. 10-1000 NTU:
are obtained by caIculating the ratio of the 90° angle ± 2 per cent of the measured value.
scattered light measured to the sum of the components of - Calibration: with 4 reference suspensions of formazin in
forward scattered and transmitted light values. In ratio the range of interest. Reference suspensions described in
turbidimetry the infiuence of stray light becomes negligible. this chapter or suitable reference standards calibrated
Nephelometers are used for measurements of the degree of against the primary reference suspensions may b~ used.
opalescence of colourIess liquids .
- Stray light: this is a significant source of error in low level
Measurements of reference suspensions I-IV with a ratio turbidimetric measurement; stray light reaches the
turbidimeter show a linear relationship between the detector of an optical system, but do es not come from the
concentrations and measured NTU values (se e sample; < 0.15 NTU for the range 0-10 NTU, < 0.5
Table 2.2.1.-2) . Reference suspensions I-IV (Ph. Eur.) may NTU for the range 10-1000 NTU.
be used as calibrators for the instrumento
Instruments complying with the aboye characteristics and
verified using the reference suspensions described under
Table 2.2.1.-2
Visual method may be used instead of visual examination for
Formazin suspensions Opalescent values (NTU) determination of compliance with monograph requirements.
Reference suspension 1 3 Instruments with range or resolution, accuracy and
Reference suspension II 6 repeatability capabilities other than those mentioned aboye
may be used provided they are sufficiently validated and are
Reference suspension 111 18
capable for the intended use. The test methodology íor the
Reference suspension IV 30 specific substance/product to be analysed must alsO be
Standard of opalescence 60 validated to demonstrate its analytical capability.
The instrument and methodology should be consistent with
Primary opalescent suspension 4000
the attributes of the product to be tested.
Instrumental determination of opalescence

Requirements in monographs are expressed in terms of the
visual examination method with the defined reference
suspensions. Instrumental methods may also be used for
determining compliance with monograph requirements once
the suitability of the instrument as described below has been
established and calibration with reference suspensions I-IV
and with water Ror the solvent used has been performed.
Apparatus Ratio turbidimeters or nephelometers with
selectable ratio application use as light source a tungsten
lamp with spectral sensitivity at about 550 nm operating at a
2014 Appendix IV B V-A233
and 975 mL of water R and dilute to 1000.0 mL with the

B. Colour of Solution same mixture. Titrate and adjust the solution to contain
(Ph. Eur. method 2.2.2) 62.4 mg of CuS04,5H 2 0 per millilitre by adding the same
The examination of the degree of coloration of liquids in the acidic mixture.
range brown-yellow-red is carried out by one of the Titration. Place in a 250 mL conical fiask fined with a
2 methods below, as prescribed in the monograph. ground-glass stopper, 10.0 mL of the solution, 50 mL of
A solution is colourless if it has the appearance of water R or water R, 12 mL of dilute aeetie acid R and 3 g of potassium
the solvent or is not more intensely coloured than reference iodide R. Titrate the liberated iodine with 0. 1 M sodium
solution B9' thiosulfate, using 0.5 mL of stal'eh solution R, added towards
the end of the titration, as indicator. The end-point is
Method 1 reached when the solution shows a slight pale brown colour.
Using identical tubes of colourless, transparent, neutral glass 1 mL of 0.1 M sodium thiosulfate is equivalent to 24.97 mg of
of 12 mm external diameter, compare 2.0 mL of the liquid CuS04,5H 2 0.
to be examined with 2.0 mL of water R or of the solvent or
Standard solutions
of the reference solution esee Tables of reference solutions)
prescribed in dle monograph. Compare the colours in Using the 3 primary solutions, prepare the 5 standard
diffused daylight, viewing horizontally against a white solutions as follows:
background.
Table 2.2.2.-1
Method 11
Volurne in rnillilitres
Using identical tubes of colourless, transparent, neutral glass
with a fiat base and an internal diameter of 15 mm to Standard solution Yellow Red Blue Hydrochloric acid
solution solution solution (10 ¡UL HCI)
25 mm, compare the liquid to be examined with water R or
8 (brown) 3.0 3.0 2.4 1.6
the solvent or the reference solution ese e Tables of reference
solutions) prescribed in the monograph, the depth of the 8Y (brownish-yellow) 2.4 LO 0.4 6.2
layer being 40 mm. Compare the colours in diffused daylight, Y (yellow) 2.4 0.6 0.0 7.0
viewing vertically against a white background.
GY (greenish-yellow) 9.6 0.2 0.2 0.0
Reagents R (red) 1.0 2.0 0.0 7.0
Primary solutions
Yellow solution Dissolve 46 g of fenie ehlonde R in about
900 mL of a mixture of 25 mL of hydl'oehlol'ie acid R and Reference solutions for Methods 1 and II
975 mL of water R and dilute to 1000.0 mL with the same Using the 5 standard solutions, prepare the following
mixture. Titrate and adjust the solution to contain 45.0 mg reference solutions.
of FeCI 3,6H zO per millilitre by adding the same acidic
mixture. Protect the solution from light. Table 2.2.2.-2. - Reference solutions B
Titration. Place in a 250 mL conical fiask fined with a
Volurnes in rnillilitres
ground-glass stopper, 10.0 mL of the solution, 15 mL of
water R, 5 mL of hydroehlone aeid R and 4 g of potassium Reference Standard solution B Hydrochloric acid
iodide R, close the fiask, allow to stand in the dark for 15 min solution (lO ¡UL HCI)
and add 100 mL of water R. Titrate the liberated iodine with 8, 75.0 25.0
0.1 M sodiwn thiosulfate, using 0.5 mL of stareh solution R, 8, 50.0 50.0
added towards the end of the titration, as indicator. 8, 37.5 62.5
1 mL of 0.1 M sodium thiosulfate is equivalent to 27.03 mg of
8. 25.0 75.0
FeCI 3 ,6H zO. .
Red solution Dissolve 60 g of eobalt ehlonde R in about 8s 12.5 87.5
900 mL of a mixture of 25 mL of hydroehlone acid R and 8, 5.0 95.0
975 mL of water R and dilute to 1000.0 mL with the same 8, 2.5 97.5
mixture. Titrate and adjust the solution to contain 59 .5 mg
of CoCI 2 ,6H zO per millilitre by adding the same acidic 8, 1.5 98.5
mixture. 8, LO 99.0
Titration. Place in a 250 mL conical fiask fined with a
ground-glass stopper, 5.0 mL of the solution, 5 mL of dilute
hydrogen peroxide solution R and 10 mL of a 300 gIL solution
of sodium hydroxide R. Boil gently for 10 min, allow to cool
and add 60 mL of dilute sulfurie acid R and 2 g of potassium
iodide R. Close the fiask and dissolve the precipitate by
shaking gently. Titrate the liberated iodine with 0.1 M sodium
thiosulfate, using 0.5 mL of stareh solurion R, added towards
the end of the titration, as indicator. The end-point is
reached when the solution turns pink.
1 mL of 0.1 M sodium thiosulfate is equivalent to 23.79 mg of
CoCl z,6H 2 0.
Blue primary solution Dissolve 63 g of eopper sulfate R in
about 900 mL of a mixture of 25 mL of hydroehlO1'ie aeid R
V-A234 Appendix IV B 2014
Table 2.2.2.-3. - Reference solutions BY Storage

For Method l, the reference solutions may be stored in
Volumes in millilitres
sealed tubes of colourless, transparent, neutral glass af
Reference Standard solution BY Hydrocbloric acid 12 mm external diameter, protected fram light.
solution (lO gIL Hel)
BY¡ 100.0 0.0
For Method II, prepare the reference solutions immediately
before use from the standard solutions.
BY, 75.0 25.0
BY, 50.0 50.0
BY, 25.0 75.0
BY, 12.5 87.5
BY, 5.0 95.0
BY, 2.5 97.5
Table 2.2.2.-4. - Reference solutions Y
Reference Standar~ solution Y Hydrocbloric acid
solution (lO glLHel)
YI 100.0 0.0
Y, 75.0 25.0
Y, 50.0 50.0
Y, 25.0 75.0
Ys 12.5 87.5
Y, 5.0 95.0
Y, 2.5 97.5
Table 2.2.2.-5. - Reference solutions GY
Reference Standard solution GY Hydrochloric acid
solution (lO giL Hel)
GY I 25.0 75.0
GY, 15.0 85.0
GY, 8.5 91.5
GY, 5.0 95.0
GYs 3.0 97.0
GY, 1.5 98.5
GY, 0.75 99.25
Table 2.2.2.-6. - Reference solutions R

Reference Standard solution R Hydrocbloric acid

solution (lO giL Hel)
R¡ 100.0 0.0
R, 75.0 25.0
R, 50.0 50.0
R, 37.5 62.5
Rs 25.0 75.0
R, 12.5 87.5
R, 5.0 95.0
2014 Appendix V A V -A235
Schedule Range Graduated Diameter Overall

Appendix V mark of stem length
a[ each
oC mm
A. Determination of Melting Point (max)
mm
Method 1
SA 5SC/80 -10 ro 55 0.5° 5.5 to 8 200
(Ph. Eur. methad 2.2.14) SA 105C/80 45 [O 105 0.5° 5.5 ro 8 200
The melting point detennined by the capillary method is the SA 155C/80 95 (O 155 0.5° 5.5 (O 8 200
temperature at which the last solid particle of a compact SA 205C/80 145 (O 205 0.5° 5.5 ro 8 200
column of a substance in a tube passes into the liquid phase. SA 225C/80 195 (O 255 0.50 5.5 ro 8 200
When prescribed in the monograph, the same apparatus and SA 305C/80 245 (O 305 0.50 5.5 ro 8 200
method are used for the detennination of other factors, such SA 360C/80 295 ro 360 0.5 0 5.5 ro 8 200
as meniscus formation or melting range, that characterise the
melting behaviour of a substance.
Apparatus The apparatus consists of: (d) Thin-walled capillary glass tubes of hard glass, closed at
- a suitable glass vessel containing a liquid bath (for one end, with a wall thickness ofO .10 to 0.15 mm, at
example, water, liquid paraffin or silicone oil) and fined least 12 cm in length and of intemal diameter 0.9 to
with a suitable means of heating, 1.1 mm. The tubes should preferably be kept sealed at
both ends and cut as required.
- a suitable means of stirring, ensuring uniforrnity of
temperature within the bath, Method Dry a small quantity of the finely powdered
substance at a temperature considerably below its melting
- a suitable thennometer with graduation at not more than
point or at a pressure of 2 kPa over a suitable desiccant,
0.5 oC intervals and provided with an immersion mark.
unless otherwise directed. Transfer a portion 10 a dry
The range of the thennometer is not more than ·100 oC,
capillary tube and pack the powder by tapping on a hard
- alkali-free hard-glass capillary tubes of internal diameter surface so as to form a tightly packed column 4 10 6 mm in
0.9 mm 10 1.1 mm with a wall 0.10 mm to 0.15 mm height. Heat a suitable liquid in the heating vessel and
thick and sealed at one end. regulate the rate of rise of temperature, prior 10 the
Method Unless otherwise prescribed, dry the finely D
introduction of the capillary tube, to 3 per minute, unless
powdered substance in vacuo and over anhydraus silica gel R otherwise directed, stirring constantly. When the temperature
for 24 h. Introduce a sufficient quantity into a capillary tube reaches 10° below the lowest figure of the range for the
to give a compact column 4 mm to 6 mm in height. Raise substance being tested, adjust the height of the thermometer
the temperature of the bath 10 about 10 °C below the so that the irnmersion mark is at the level of the surface of
presumed melting point and then adjust the rate of heating the liquid and insert the capillary tube so that the closed end
10 about 1 oC/mino When the temperature is 5 oC below the is near the middle of the bulb of the thermometer. Note the
presumed melting point, correctly introduce the capillary temperature at which the liquefaction of the substance
tube into the instrumento For the apparatus described aboye, occurs, which is indicated by the formation of a definite
irnmerse the capillary tube so that the closed end is near the meniscus or, for substances that decompose, the temperature
centre of the bulb of the thermometer, the immersion mark at which frothing begins. Correct the observed temperature
of which is at the level of the surface of the liquido Record for any error in the calibration of the thermometer and for
the temperature at which the last particle passes into the the differen¡:;e, if any, between the temperature of the
liquid phase. emergent stem of the thermometer and the temperature of
Calibration 01 the apparatus The apparatus may be the emergent stem under the conditions of standardisation of
calibrated using melting point reference substances such as the thermometer. The temperature of the emergent stem is
those of the World Health Organisation or other appropriate determined by placing the bulb of a second thermometer in
substances . contact with the emergent stem at a point approximately
midway along the mercury thread in the emergent stem.
Method 11 The correction 10 be applied is given by the fol!owing
(No Ph. Eur. methad) equation:
Apparatus te = 0.000 16n(ts - td)
(a) A glass heating vessel of suitable construction and where te correction 10 be added to the observed
capacity containing one of the following, or another temperature of the melting point,
suitable liquid, 10 a height of not less than 14 cm. ts mean temperature of the emergent column
when standardised,
(i) A liquid paraffin of sufficiently high boiling point.
mean temperature of the emergent column at
(ii) A silicone fluid of sufficiently high boiling point. the observed melting point,
(iii) Water. n number of oC over which the exposed column
(b) A suitable stirring device capable of rapidly mixing the extends.
liquido The corrected temperature is regarded as the melting point
(e) An accurately standardised thennometer suitable for the of the substance. When the melting point in the monograph
substance being examined complying with the is expressed as a range, the melting point of the substance
requirements of British Standard 1365: 1990 being tested must fal! within that range.
(Specification for short-range short-stem thennometers)
for thennometers designated by one of the following
Schedule Marks.
V-A236 Appendix V A 2014
Method III grooved stopper through which the thermometer passes.

(Ph. Eur. method 2.2.17) The opening of the cup is placed about 15 mm from the
The drop point is the temperature at which the first drop of bottom of the test-tube. The whole device is immersed in a
the melting substance to be examined falls from a cup under beaker with a capacity of about 1 litre, filled with water.
defined conditions. The bottom of the test-tube is placed about 25 mm from the
bottom of the beaker. The water level reaches the upper part
When a monograph do es not specifY the method to be used,
of sheath A. A stirrer is used to ensure that the temperature
method A is applied. Any change from method A to
of the water remains uniformo
method B is validated.
Method Prepare the substance to be examined according
Method A to the prescriptions of the monograph. Fill the cup to the
brim with the substance to be examined. Remove the excess
Apparatus The apparatus (see Figure 2.2.17.-1 ) consists
substance at the 2 ends of the cup with a spatula. When
of 2 metal sheaths (A and B) screwed together. Sheath A is
sheaths A and B have been assembled, press the cup into its
fixed to a mercury thermometer. A metal cup is loosely fixed
housing in sheath B until it touches the supports. Remove
to the lower part of sheath B by means of 2 tightening
with a spatula the substance pushed out by the thermometer.
bands. Fixed supports 2 mm long determine the exact
Place the apparatus in the water-bath as described above.
position of the cup, and in addition are used to centre the
Heat the water-bath and, when the temperature is at about
thermometer. A hole pierced in the wall of sheath B is used
10 oC below the presumed drop point, adjust the heating
to balance the pressure. The draining surface of the cup
rate to about 1 oC/mino Note the temperature at the fall of
must be fiat and the edges of the outfiow orifice must be at
the first drop . Carry out at least 3 determinations, each time
right angles to it. The lower part of the mercury
with a fresh sample of the substance. The difference between
thermometer has the form and size shown in the figure;
the readings must not exceed 3 oc. The mean of 3 readings
it covers a range from O oC to 110 oC and on its scale a
is the drop point of the substance.
distance of 1 mm represents a difference of 1 oc.
The mercury reservoir of the thermometer has a diameter of
Method B - Automated method
3.5 ± 0.2 mm and a height oL6.0 ± 0.3 mm.
The apparatus is placed in the axis of a test-tube about Apparatus The apparatus (se e Figure 2.2.17 .-2) consists
200 mm long and with an external diameter of about of a cartridge assembly comprising a cup holder into which
40 mm. Ir is fixed to the test-tube by means of a laterally the sample cup containing the sample is loosely fixed, and a
collector sleeve with a horizontal light slit, which is fixed
below the cup. This assembly is placed in a heating block.
The block is a metal cylinder with a cylindrical hole along its
f! f+ vertical axis into which the cartridge assembly is placed.
There is another, narrower cylindrical vertical hole in which
a temperature sensor sits. This is positioned level with the
~ sample cup. The heating block is surrounded by an electrical
heating elemento Below the heating block a lamp is mounted
such that a beam of light shines rhrough the light slit in the
collector sleeve, and onto a photo-sensor mounted opposite.
The heating block is capable of being maintained at a
10 precise, pre-defined temperature by the heating element, and
12.5 of being heated at a slow and steady, pre-defined rate after
an initial isothermal periodo
Method Melt the substance to be examined and introduce
1111111
-+------- A ------~
it into the sample cup according to the prescriptions of the
monograph, then proceed as follows or according to the
manufacturer's instructions. Remove the excess substance at
the 2 ends of the cup with a spatula. Condition the sample
at the temperature and for the time prescribed in the
....
<J)
monograph before making the measurement. Press the cup
into the cup holder, and then press the collector sleeve onto
the cup. Place the c3:rtridge assembly in the heating block.
Set the instrument to the initial isothermal conditions and
rate for subsequent heating as described in the monograph of
the substance to be examined. Start the temperature
11 programme. When the first drop of molten sample falls
__~5____.1 +---- through the hole at the bottom of the sample cup,
interrupting the light beam, the signal from the photo-sensor
causes the temperature of the heating block to be recorded
A. upper metal sheath D. fixed supports automatically.
B. lower metal sheath E. tightening bands
C. pressure-balancing hole F. metal sample cup
Figure 2.2.17.-1. - Apparafus for the determination

of drop poinf
2014 Appendix V A V-A237
TI mean drop point temperature of 3 samples, in oC,

E F compensation for the difference in temperature
between the sample and the point in the heating
F block where the temperature is measured; this will
vary depending upon the design of the automatic
drop point instrument and is provided by the
manufacturero
A G Taking into account the drop point (Ta) of the certified
reference material, the accuracy of the temperature scale is
satisfactory if IT2 - Ta l is not greater than 0.3 oC.
B MethodIV
For certain substances, the following method is used to
determine the melting point (also referred to as slip point
and rising melting point when determined by this method).
Use glass capillary tubes open at both ends, about 80 mm
e long, having an external diameter of 1.4 mm to 1.5 mm and
an internal diameter of 1.0 mm to 1.2 mm.
Introduce into each of 5 capillary tubes a sufficient amount
of the substance, previously treated as described, to form in
each tube a column about 10 mm high and allow the tubes
to stand for the appropriate time and at the prescribed
A. cup holder F. heating element temperature.
B. heating block G. sample cup Unless otherwise prescribed, substances with a waxy
c. Iight source H. photo-sensor
consistency are carefully and completely melted on a water-
bath before introduction into the capillary tubes. Allow the
D.light slit J. collector sleeve tubes to stand at 2-8 oC for 2 h.
E. cartridge assembly K. temperature probe Attach one of the tubes to a thermometer graduated in
0.5 oC so that the substance is close to the bulb of the
Figure 2.2.17.-2. - Example of automated drop point apparatus thermometer. Introduce the thermometer with the attached
tube into a beaker so that the distance between the bottom of
Calibration Use the apparatus according to the the beaker and the lower part of the bulb of the thermometer
manufacturer's instructions and carry out the prescribed is 1 cm. Fill the beaker with water to a depth of 5 cm.
calibrations and system performance tests at regular intervals, lncrease the temperature of the water gradually at arate of
depending on the use of the apparatus and the substances to 1 oC/mino
be examined. Benzoic acid and benzophenone are usually The temperature at which the substance begins to rise in the
used as certified reference materials. Other materials may be capillary tube is regarded as the melting point.
used provided they show no polymorphism. Proceed as
Repeat the operation with the other 4 capillary tubes and
follows or according to the manufacturer's instructions.
Prepare 3 sample cups for each of the 2" certified reference calculate the result as the mean of the 5 readings.
materials. Place the sample cups on a clean surface. lnto Method V
each sample cup, introduce a small quantity of the sample
and press it down with a rod (diameter about 4.5 mm).
The instantaneous melting point is calculated using the
Check that the opening is completely filled. Fill the sample
expression:
cup about half full and compact the sample with a rod
(diameter about 9 mm). Fill the sample cup completely and
compact, adding more sample and compacting again if
necessary, until the sample cup is completely full.
in which tI is tlÍe first temperature and t2 the second
Temperature prograrnme for benzoic acid: start temperature read under the conditions stated below.
temperature = 118.0 oC; heating rate = 0.2 °C/min;
Apparatus The apparatus consists of a metal block
end temperature = 126.0 oc. After inserting the cup at
resistant to the substance to be examined, of good heat-
118 oC, a waiting time of 30 s is set before heating starts.
conducting capacity, such as brass, with a carefully polished
Temperature programme for benzophenone: start plane upper surface. The block is uniforrnly heated
temperature = 44.0 oC; heating rate = 0.2 °C/min; throughout its mass by means of a micro-adjustable gas
end temperature = 56.0 oC. After inserting the cup at 44°C, heater or an electric heating device with fine adjustment.
a waiting time of 30 s is set before heating starts. The block has a cylindrical cavity, wide enough to
Check the 3 single results:the test is valid if the 3 results are accomodate a thermometer, which should be maintained
within 0.3 oC of the mean value. with the mercury column in the same position during the
Calculate the corrected mean temperature (1)2 using the calibration of the apparatus and the determination of the
following expression: melting point of the substance to be examined.
The cylindrical cavity is parallel to the upper polished surface
of the block and about 3 mm from it. The apparatus is
calibrated using appropriate substances of known melting
point.
V-A238 Appendix V A 2014
Method Heat the block at a suitably rapid rate to a Place the capillary tube in the heating block with the closed
temperature about 10 oC below the presumed melting end downwards. Start the temperature programme. When
temperature, then adjust the heating rate to about 1 oC/mino the substance starts melting, it changes its appearance in the
At regular intervals drop a few particles of powdered and, capillary tube. As a result, the temperature of the heating
where appropriate, dried substance, prepared as for the block is recorded automatically following the signal changes
capillary tube method, onto the block in the vicinity of the from the photosensor due to light transmission (mode A,
thermometer bulb, cleaning the surface after each test. Figure 2.2.60.-1), or following image processing (mode B,
Record the temperature t[ at which the substance melts Figure 2.2.60.-2).
instantaneously for the first time in contact with the metal. Carry out the test on 2 other samples and calculate the mean
Stop the heating. During cooling drop a few particles of the value of the 3 results.
substance at regular intervals on the block, cleaning the Calibration The temperature scale of the apparatus is
surface after each test. Record the temperature t2 at which checked periodically by measuring the melting point of
the substance ceases to melt instantaneously when it comes certified reference materials. Use capillary tubes having the
in contact with the metal same dimensions as those used for the determination of the
Calibration 01 the apparatus The apparatus may be melting point (see Apparatus).
calibrated using melting point reference substances such as Prepare 3 capillary tubes for each of at least 2 certified
those of the World Health Organisation or other appropriate reference materials. Carry out the test and calculate the mean
substances. value of the 3 results for each material.
Method VI System suitability In addition to the calibration, carry out
a verification, before the measurements, using a suitable
certified reference material whose melting point is close to
This chapter describes the measurement of melting point by
that expected for the substance to be examined.
the capillary method using an instrumental method of
determination. Prepare 3 capillary tubes. Carry out the test and calculate the
mean value of the 3 results.
Apparatus There are 2 modes of automatic observation
arrangements: The mean value is within the tolerance given on the
certificate supplied with the certified reference material.
- mode A: by light transmission through the capillary tube
loaded with the sample;
- mode B: by light being reftected from the sample in the
capillary tube.
In both modes, the capillary tube sits in a hollow of a metal
block, which is heated electrically and controlled by a A
temperature sensor placed in another hollow of the metal
block. The heating block is capable of being maintained
E
accurately at a pre-defined temperature (± 0.1 oC) by the
heating element, and of being heated at a slow and steady
rate of 1 °C/min, after an initial isothermal periodo
In mode A, a beam of light shines through a horizontal
hollow and crosses the capillary tube. A sensor detects the
beam at the end of the cylindrical hole after the capillary
tube.
In mode B, a beam of light illuminates the capillary tube
F
from the front and the sensor records the image.
Sorne apparatuses allow for the visual determination of the é)
melting point.
The temperature at which the sensor signal first leaves its e
initial value is defined as the beginning of melting, and the
temperature at which the sensor signal reaches its final value o
is defined as the end of melting, or the melting point.
Use glass capillary tubes that are open at one end, about
lOO mm long, with an external diameter of 1.3-1.5 mm and
an internal diameter of 0.8-1.3 mm. The wall thickness of
the tube is 0.1-0.3 mm.
Sorne apparatuses allow for the determination of the melting E
point on more than 1 capillary tube.
Method Introduce iÍno the capillary tube a sufficient A. glass capillary tube D. temperature sensor
amount of the substance to be examined, previously treated
as described in the monograph, to form in each tube a B. sample E. heating block
compact column about 4 mm high, and allow the tubes to c. photosensor F. light source
stand for the appropriate time at the prescribed temperature.
Proceed as follows or according to the manufacturer's
Figure 2.2.60.-1. - Mode A: transmission
instructions. Heat the heating block until the temperature is
about 5 oC below the expected melting point.
2014 Appendix ve V-A239
1-
1--
1-
B
~
;: 1\¡-.!+18;:"'"
D e
t 1+ 25 4
1+-- 40 ---->-
Figure 2.2.18.-1. - Apparatus for the determination of

freezing point
Method Place in the inner tube sufficient quantity of the

liquid or previously melted substance to be examined, to
E coyer the thermometer bulb and determine the approximate
freezing point by cooling rapidly. Place the inner tube in a
bath about 5 ce aboye the approximate freezing point until
A. glass capillary tube E. heating block all but the last traces of crystals are melted. Fill the beaker
B. sample F. light source with water or a saturated solution of sodium chloride, at a
temperature about 5 oC lower than the expected freezing
C. imagesensor G. transparent plate
point, insert the inner tube into the outer tube, ensuring that
D. temperature sensor sorne seed crystals are present, and stir thoroughly until
solidification takes place. Note the highest temperature
Figure 2.2.60.-2. - Mode B: reflexion observed during solidification.
C. Determination 01 Distillation Range

B. Determination 01 Freezing Point (Ph . Eur. methad 2.2.11)
The distillation range is the temperature interval, corrected
(Ph. Eur. methad 2.2.18) for a pressure of 10l.3 kPa (760 Torr), within whieh a
The freezing point is the maximum temperature occurring liquid, or a speeified fraetion of a liquid, distils in the
during the solidification of a supercooled liquido following conditions.
Apparatus The apparatus (see Figure 2.2 .18.-1) consists Apparatus The apparatus (se e Figure 2.2.1l.-1) consists
of a test-tube about 25 mm in diameter and 150 mm long of a distillation flask (A), a straight tube eondenser (B)
placed inside a test-tube about 40 mm in diameter and whieh fits on to the side arm of the flask and a plain-bend
160 mm long. The inner tube is closed by a stopper which adaptor (C) attached to the end of the condenser. The lower
carries a thermometer about 175 mm long and graduated in end of the eondenser may, aIternatiyely, be bent to replaee
0.2 oC fixed so that the bulb is about 15 mm aboye the the adaptor. A thermometer is inserted in the neek of the
bottom of the tube. The stopper has a hole allowing the flask so that the upper end of the mercury reservoir is 5 mm
passage of the stem of a stirrer made from a glass rod or lower than the junetion of the lower wall of the lateral tube.
other suitable material formed at one end into a loop of The thermometer is graduated at 0.2 ce intervals and the
about 18 mm oyerall diameter at right angles to the rod. scale coyers a range of about 50 oc. During the
The inner tube with its jacket is supported centrally in a determination, the flask, including its neek, is proteeted from
1 litre beaker containing a suitable cooling liquid to within draughts by a suitable sereen.
20 mm of the top o A thermometer is supported in the
cooling bath.
V-A240 Appendix VD 2014
Figure 2.2.11.-1. - Apparatus far the determinatian af distillatian range

Dimensians in millimelres
Method Place in the flask (A) 50.0 mL of the liquid to be

examined and a few pieces of porous material. Collect the
D. Determination of Boiling Point
distillate in a 50 mL cylinder graduated in 1 mL. Cooling by (Ph. Eur. methad 2.2.12)
circulating water is essential for liquids distilling below The boiling point is the corrected temperature at which the
150 oC. Heat the flask so that boiling is rapidly achieved and vapour pressure of a liquid is equal to 101.3 kPa.
note the temperature at which the first drop of distillate falls Apparatus The apparatus is that used for Distillation
into the cylinder. Adjust the heating to give a regular rate of Range (2.2.11) with the exception that the thermometer is
distillation of 2-3 mUmin and note the temperature when inserted in the neck of the flask so that the lower end of the
the whole or the prescribed fraction of the liquid, measured mercury reservoir is level with the lower end of me neck of
at 20 oC, has distilled. the distillation flask and that the flask is placed on a plate of
Correct the observed temperarures for barometric pressure by isolating material pierced by a hole 35 mm in diameter.
means of the formula : Method Place in the flask (A ) 20 mL of the liquid to be
examined and a few pieces of porous material. Heat the flask
tl = t2 + k (101.3 - b) so that boiling is rapidly achieved and record the
temperature at which liquid runs from the side-arm into the
ti the corrected temperature, condenser.
t2 the observed temperature, at the barometric pressure Correct the observed temperature for barometric pressure by
b, means of the formula:
k the correction factor taken from Table 2.2.11.-1 unless
the factor is given, tl = t2 + k (101.3 - b)
b the barometric pressure, expressed in kilopascals,
during the distillation. ti the corrected temperature,
t2 the observed temperature at barometric pressure b,
k the correction factor as shown in Table 2.2.11.-1
Table 2.2 .11.-1. - Temperature correction in relation to the
pressure under Distillation Range,
b the barometric pressure, in kilopascals, at the time of
Distillation temperature Correction factor k
the determination.
up to 100 oc 0.30
aboye 100 oc up to 140 oC 0.34
aboye 140 oC up to 190 oC 0.38
aboye 190 oC up to 240 oC 0.41
aboye 240 oC 0.45
2014 Appendix V G V -A241
units; the actual units, degree millilitres per decimetre gram

E. Determination of Refractive Index [CO).mLdm- 1g- 1j are understood.
(Ph. Eur. method 2.2.6) The conversion factor from the Intemational System to the
The refractive index of a medium with reference to air is Pharmacopoeia system is the following:
equal to the ratio of the sine of the angle of incidence of a
beam of light in air to the sine of the angle of refraction of [am]~ = [a]~ x 0.1745
the refracted beam in the given medium.
Unless otherwise prescribed, the refractive index is measured In certain cases specified in the monograph the angle of
at 20 ± 0.5 oC, with reference to the wavelength of the rotation may be measured at temperatures other than 20 cC
D-line ofsodium (A = 589.3 nm); the symbol is then nbo. and at other wavelengths.
Refractometers normally determine the critical angle. In such The polarimeter must be capable of giving readings to the
apparatus the essential part is a prism of known refractive nearest 0.01 The scale is usually checked by means of
0,
index in contact with the liquid to be examined. certified quanz plates. The linearity of the scale may be
Calibrate the, apparatus using cenified reference materials. checked by means of sucrose solutions.
When white light is used, the refractometer is provided with Method Determine the zero of the polarimeter and the
a compensating system. The apparatus gives readings angle of rotation of polarised light at the wavelength of the
accurate to at least the third decimal place and is provided D-line of sodium (A = 589.3 nm) at 20 ± 0.5 oC, unless
with a means of operation at the temperature prescribed. otherwise prescribed. Measurements may be carried out at
The thermometer is graduated at intervals of 0.5 oC or less. other temperatures only where the monograph indica tes the
temperature correction to be made to the measured optical
rotation. Determine the zero of the apparatus with the tube
closed; for liquids the zero is determined with the tube
F. Determination of Optical Rotation and empty and for solids filled with the prescribed solvent.
Specific Optical Rotation Calculate the specific optical rotation using the following
formulae.
Optical rotation is the propeny displayed by chiral substances For neat liquids:
of rotating the plane of polarisation of polarised light. 20 _ _ a_
[a ] D -
Optical rotation is considered to be positive (+) for l· pzo
dextrorotatory substances (i.e. those that rota te the plane of
polarisation in a clockwise direction) and negative (-) for For substances in solution:
laevorotatory substances.
The specific optical rotation [aml;, is the rotation, expressed 20 _ 1000a
[
in radians (rad), measured at the temperature t and at the a ]D - l. e
wavelength A given by a 1 m thickness of liquid or a solution
containing 1 kg/m 3 of optically active substance. For practical where e is the concentration of the solution in grams per litre.
reasons the specific optical rotation [aml ~ is normally Calculate the content e in grams per litre or the content c ' in
expressed in milliradians metre squared per kilogram per cent m/m of a dissolved substance using the following
(mrad·m 2 kg- 1), formulae:
The Pharmacopoeia adopts the following conventional
definitions , 1000a f 100a
c=--- e
l· [a]~o 1 . [a]~o . pzo
The angle oloptical rotation of a neat liquid is the angle of
rotation r:J., expressed in degrees CO), ofthe plane of
polarisation at the wavelength of the D-line of sodium r:J. angle of rotation in degrees read at 20 ± O.5°C,
(A = 589 .3 nm) measured at 20 oC using a layer of 1 dm; length in decimetres of the polarimeter tube,
for a solution, the method of preparation is prescrib¡;d in the Pzo density at 20 oC in grams per cubic centimetre.
monograph. For the purposes of the Pharmacopoeia, density is
replaced by relative density (2.2,5),
The specijic optical rotation [al~o of a liquid is the angle of
rotation r:J., expressed in degrees CO), of the plane of
polarisation at the wavelength of the D-line of sodium
(A = 589.3 nm) measured at 20 oC in the liquid substance to
be examined, calculated with reference to a layer of 1 dm G. Determination of Weight per Millilitre,
and divided by the density expressed in grams per cubic Density, Relative Density and Apparent
centimetre.
The specific optical rotation [alba of a substance in solution is Weight per millilitre
the angle of rotation r:J., expressed in degrees (0), of the plane (No Ph. Eur. method)
of polarisation at the wavelength of the D-line of sodium The weight per m¡llilitre of a liquid is the weight in g of 1 mL
(A = 589.3 nm) measured at 20 oC in a solution of the of a liquid when weighed in air at 20°, unless otherwise
substance to be examined and ca1culated with reference to a specified in the monograph.
layer of 1 dm containing 1 g/roL of the substance , The weight per millilitre is determined by dividing the weight
The specific optical rotation of a substance in solution is in air, expressed in g, of the quantity of liquid that fills a
always expressed with reference to a given solvent and pycnometer at the specified temperature by the capacity,
concentration. expressed in mL, of the pycnometer at the same temperature.
In the conventional system adopted by the Pharmacopoeia The capacity of the pycnometer is ascertained from the
the specific optical rotation is expressed by its value without weight in air, expressed in g, of the quantity of water required
V-A242 Appendix V G 2014
to fill the pycnometer at that temperature. The weight of a (solids), a hydrometer (liquids) or a digital density meter
litre of water at specified temperatures when weighed against with an oscillating transducer (liquids and gases). When the
brass weights in air of density 0.0012 g per mL is given in determination is made by weighing, the buoyancy of air is
the following tableo Ordinary deviaúons in the density of air disregarded, which may introduce an error of 1 unit in the
from the above value, here taken as the mean, do not affect 3rd decimal place. When using a density meter, the buoyancy
the result of a determination in the significant figures of air has no influence.
prescribed for Pharmacopoeial substances. Oscillating transducer density meter The apparatus
consists of:
Temperature Weíght ofa - a U-shaped tube, usually of borosilicate glass, which
litre of water contains the liquid to be examined;
oC g - a magneto-electrical or piezo-electrical excitation system
20 997.18 that causes the tube to oscillate as a cantilever oscillator at
25 996.02 a characteristic frequency depending on the density of the
30 994.62 liquid to be examined;
- a means of measuring the oscillaúon period (1), which
may be converted by the apparatus to give a direct
reading of density, or used to calculate density using the
Density
constants A and B described below.
(No Ph. Eur. methad)
The resonant frequency (j) is a funcúon of the spring
The density, P20, of a substance is the ratio of its mass to its constant (e) and the mas s (m) of the system:
volume at 20°. It is expressed in kg m- 3 .
The density is determined by dividing the weight in air of the
quantity of the liquid being examined that fills a pycnometer
at 20° by the weight in, air of water required to fill the
pycnometer after making allowance for the thrust of the airo Hence:
The density is calculated from the expression
998.2(M¡ + A)
P20 = M +A
2 M = mass of the tube,
V = inner volume of the tube.
where MI weight in air (apparent mass) in grams of
Introduction of 2 constants A = e /(4n 2 x V) and B = M /V,
the substance being examined,
leads to the classical equation for the oscillaúng transducer:
weight in air (apparent mass) in grams of
water, 2
P=A X T - B
A the correction factor for the thrust of the
air,0.0012M2
the density of water at 20° in kg m- 3. The constants A and B are determined by operaúng the
998 .2
instrument with the U-tube filled with 2 different samples of
In most cases, the correction for the thrust of the air may be known density, for example, degassed water R and airo
disregarded. Control measurements are made daily using degassed
water R. The results displayed for the control measurement
Relative density using degassed water R shall not deviate from the reference
(Ph. Eur. methad 2.2.5) value (P20 = 0.998203 g·cm- 3, dig = 1.000000) by more
The relative density d:; of a substance is the ratio of the mass than its specified error. For example, an instrument specified
of a certain volume of a substance at temperature tI to the to ± 0.0001 g·cm- 3 shall display 0.9982 ± 0.0001 g·cm- 3
mass of an equal volume of water at temperature t2. in order to be suitable for further measurement. Otherwise a
Unless otherwise indicated, the relative density dig is used. re-adjustment is necessary. Calibration with certified
Relative density is also commonly expressed as d¡o. Density reference material s is carried out regularly. Measurements are
P20, defined as the mass of a unit volume of the substance at made using the same procedure as for calibration. The liquid
20 oC may also be used, expressed in kilograms per cubic to be examined is equilibrated in a thermostat at 20 oC
metre or grams per cubic centimetre (1 kg·m- 3 = 10- 3 before introduction into the tube, if necessary, to avoid the
g·cm - 3). These quantities are related by the following formation of bubbles and to reduce the time required for
equations where dens!ty is expressed in grams per cubic measurement.
centimetre: Factors affecting accuracy include:
- temperature uniforrnity throughout the tube,
, 20 20
P20 = 0.998203 X d 20 or d 20 = 1.00180 X P20 - non-linearity over a range of density,
- parasitic resonant effects,
20
P20 = 0.999972 X d 4 or d¡O = 1.00003 X P20 - viscosity, whereby solutions with a higher viscosity than
the calibrant have a density that is apparently higher than
the true value.
The effects of non-linearity and viscosity may be avoided by
Relative density or density are measured with the precision to using calibrants that have density and viscosity close to those
the number of decimals prescribed in the monograph using a of the liquid to be examined (± 5 per cent for density,
density botde (solids or liquids), a hydrostaúc balance ± 50 per cent for viscosity). The density meter may have
2014 Appendix V H V-A243
functions for automatic viscosity correction and for correction The kinematic viscosity is usually expressed in square
of errors arising from temperature changes and non-linearity. millimetres per second.
Precision is a function of the repeatability and stability of the A capillary visco meter may be used for determining the
oscillator frequency, which is dependent on the stability of viscosity of Newtonian liquids and a rotating viscometer for
the volume, mass and spring constant of the cell. determining the viscosity of Newtonian and non-Newtonian
Density meters are able to achieve measurements with an liquids. Other viscometers may be used provided that the
error of the order of 1 x 10- 3 g·cm -3 to 1 X 10- 5 g.cm- 3 accuracy and precision is not les s than that obtained with the
and a repeatability of 1 x 10- 4 g·cm- 3 to 1 x 10- 6 viscometers described below.
g.cm- 3 .
Method 1
Apparent density (No Ph. Eur methad)
(No Ph. Eur. method) Apparatus
The term 'Apparent density' is used in the monographs for
The apparatus consists of a glass U-tube visco meter
Dilute Ethanols, Industrial Methylated Spirit and Industrial
(Fig.5H-l ) made of clear borosilicate glass and constructed
Methylated Spirit (Ketone-free). It is defined as weight in air
in accordance with the dimensions shown in the figure and
per unit volume and expressed in kg m- 3 . It is named
in Table 5H- 1. The monograph states the size of viscometer
'density' in the Laboratory Alcohol Table for Laboratory Use
to be used .
(HM Customs and Excise 1979).
The apparent density is calculated from the following Method
expression: Fill the viscometer with the liquid being examined through
apparent density = 997.2 x d~g tube L to slightly aboye the mark G, using a long pipette to
minimise wetting the tube aboye the mark. Place the tube
where d~gis the relative density of the substance being
vertically in a water bath and when it has attained the
examined and 997.2 is the weight in air in kg of 1 cubic
specified temperature, adjust the volume of the liquid so that
metre of water.
the bottom of the meniscus settles at the mark G. Adjust the
liquid to a point about 5 mm aboye the mark E. After
releasing pressure or suction, measure the time taken for the
H. Viscosity bottom of the meniscus to fall from the top edge of mark E
to the top edge of mark F .
(Ph. Eur. melhod 2.2.8)
The dynamic viscosity or viscasity coefficient r¡ is the tangential Calculate, as required, either the kinematic viscasity (v) in
force per unit surface, known as shearing stress T and square millimetres per second (mm 2 S- I) from the
expressed in pascals, necessary to move, parallel to the sliding expression:
plane, a layer of liquid of 1 square metre at a rate (v) of 1 v = Kt,
metre per second relative to a parallellayer at a distance (x)
of 1 metre. or the dynamic viscosity (r¡) in millipascal seconds (mPa s)
The ratio dvldx is a speed gradient giving the rate of shear D from the expression:
expressed in reciprocal seconds (S- I), so that 1) = , ID. r¡ = Kpt,
The unit of dynamic viscosity is the pascal second (Pa·s).
The most commonly used submultiple is the millipascal
where t time in seconds for the meniscus to fall from E
second (mPa·s).
to F,
The kinematic viscosity v, expressed in square metres per p mass/volume (g cm- 3) obtained by multiplying
second, is obtained by dividing the dynamic viscosity 1) by the relative density, Appendix V G, of the fluid
the density p expressed in kilograms per cubic metre, of the being examined by 0.9982.
liquid measured at the same temperature, i.e. v = r¡lp.
Table 5H- 1 U-Tube Viscometer - Dimensions
Size Nominal Kinematic Inside Outside Volume of Vertical Outside

viscometer viscosity · diameter diameterof bulbC distance diameterof
constant range ofrubeR robes 1 FtoG bulbsAand C
LandP N
mm2s-2 mm 2s- 1 mm(±2%) mm (±5%) mm mm
mm mm
----
A2 0.003 0.9 ro 3 0.50 8 ro 9 6lO7 5.0 9l±4 21 to 23
B 0 ,01 2.0 to 10 0 .71 8 to 9 6to 7 5.0 87±4 21 to 23
C 0.\13 6 to 30 0.88 8 ro 9 6 to 7 5.0 83±4 21 ro 23
D 0 .1 20 lO 100 1.40 9 lO 10 7 to 8 10.0 78±4 25 ro 27
E 0.3 60 to 300 2.00 9 to 10 7 to 8 10.0 73±4 25 to 27
F 1.0 200 to 1000 2.50 9 to 10 7roB 10.0 70±4 25 to 27
G 3.0 600 ' ro 3000 4.00 10 to 11 9 to 10 20.0 60±3 32 to 35
H 10.0 2000 ro 10,000 6.10 10 ro 11 9 to 10 20.0 50±3 32 lO 35
IUse 1 to 1.25 mm wall tubing for L, N and P.

2300 s mínimum flow time; 200 s mínimum f10w time for all other sizes.
V-A244 Appendix V H 2014
Table 2.2.9.-1
~ Size Nominal Kinematic Intemal Volume Intemal
number constant viscosity diameter of diameter
ofvisco- range oftube bulb oftube
N 75 meter R e N
rnm2·s-Z mm 2's- 1 mm mL mm
(±2 %) (±5 %)
E
0.01 3.5 to 10 0.64 5.6 2.8 to 3.2
L e lA 0.03 6 to 30 0.84 5.6 2.8 to 3.2
2 0.1 20 to 100 1.15 5.6 2.8 to 3.2
F 2A 0.3 60 to 300 1.51 5.6 2.8 to 3.2

25 300
3 1.0 200 to 1000 2.06 5.6 3.7 to 4.3
3A 3.0 600 to 3000 2.74 5.6 4.6 to 5.4

4 10 2000 to 3.70 5.6 4.6 to 5.4
G 10 000
4A 30 6000 to 4.07 5.6 5.6 to 6.4
R 120
A 30000
5 100 20000 to 6.76 5.6 6.8 to 7.5
100000
L M N
p r
Fig. SH-l
U-Tube Visco meter
¡:::
The constant (K) of the instrument is determined using the
appropriate European Pharmacopoeia reference liquid for
1(0
viscometers.
Method II (Capillary viscometer method)
~¡
ve ----
E

The determination of viscosity using a suitable capillary
viscometer is carried out at a temperature of 20 ±
0.1 oC, ~1
l'
----
F
unless otherwise prescribed. The time required for the level ,

of the liquid 10 drop from one mark 10 the other is measured
with a s1Op-watch to the nearest one-fifth of a second.
The result is valid only if two consecutive readings do not
differ by more than 1 per cent. The average of not fewer
than three readings gives the flow time of the liquid to be
o
(J) ----
R
examined.
Calculate the dynamic viscosity 11 (2.2.8) in millipascal
:
seconds using the formula:
~1
\" "-
TI = kpt B
k constant of the viscometer, expressed in square

LO
~
:---
/'
" "-
millimetres p~r second squared, o A
"<t
p density of the liquid to be examined expressed in
milligrams per cubic millimetre, obtained by
t =
multiplying its relative density (dgg) by 0.9982,
flow time, in seconds, of the liquid to be examined. ~1 '(é
The constant k is determined using a suitable visco meter Figure 2.2.9.- 1. - Suspended level viscomeler
calibration liquido
To calculate the kinematic viscosity (mm 2 s- 1), use the
The minimum flow time should be 350 s for size no. 1 and
following formula: v = kt.
200 s for all other sizes.
The determination may be carried out with an apparatus
Method Fill the visco meter through tube (L) with a
(Figure 2.2.9.-1) having the specifications described in
sufficient quantity of the liquid to be examined, previously
Table 2.2.9 .-1 1:
brought 10 20 oC unless otherwise prescribed, to fill bulb (A)
but ensuring that the leve! of liquid in bulb (E) is below the
1 The European Phannacopoeia describes the sysrem proposed by rhe exit 10 ventilation tube (M) . Immerse the visco meter in the
Internarional Organisarion jor Standardisarion (ISO). bath ofwater at 20 ± 0.1 cC, unless otherwise prescribed,
2014 Appendix V H V -A245
maintain it in the upright position and allow to stand for not where A and B are constants for the instrument and are
less than 30 min to allow the temperature to reach calculated from the following expressions:
equilibrium. Close tube (M) and raise the level of the liquid - for concentric surface:
in tube (N) up to a level about 8 mm aboye mark (E). Keep
2
the liquid at this level by closing tube (N) and opening tube A=
1R
1
+ R o2
(M). Open tube (N) and measure, with a stop-watch to the 47rh Ri R~
nearest one-fifth of a second, the time required for the level
of the liquid to drop from mark (E) to (F). - for cone-plates:
Method III (Rotating viscometer method) A=_3_

27rR3
B=~
a
(Ph. Eur. methad 2.2. 10)
The principie of the method is to measure the force acting M torque in Newton-metres acting on the cone or
on a rotor (torque) when it rotates at a constant angular cylinder surface,
velocity (rotational speed) in a liquid. Rotating viscometers úJ angular velocity in radians per second,
are used for measuring the viscosity of Newtonian R¡ radius in metres of the inner cylinder,
(shear-independent viscosity) or non-Newtonian liquids Ro radius in metres of the outer cylinder,
(shear dependent viscosity or apparent viscosity). Rotating R radius in metres of the cone,
viscometers can be divided in 2 groups, namely absolute and h height of imrnersion in metres of the inner cylinder in
relative viscometers. In absolute viscometers the flow in the the liquid medium,
measuring geometry is well defined. The measurements result a angle in radians between the flat disk and the cone,
in absolute viscosity values, which can be compared with any 1: shear stress in pascals (Pa),
other absolute values. In relative viscometers the flow in the y shear rate in reciprocal seconds (S-I)
measuring geometry is not defined. The measurements result
in relative viscosity values, which cannot be compared with ro
absolute values or other relative values if not deterrnined by
the same relative visco meter method.
Different measuring systems are available for given viscosity
ranges as well as several rotational speeds. M
Apparatus
The following types of instruments are most common.
Concenuic cylinder viscameters (absolute v iscameters)
In the concentric cylinder viscometer (coaxial double cylinder
viscometer or simply coaxial cylinder viscometer), the
viscosity is deterrnined by placing the liquid in the gap
between the inner cylinder and the outer cylinder. Viscosity
measurement can be perforrned by rotating the inner cylinder h
(Searle type viscometer) or the outer cylinder (Couette type
viscometer), as shown in Figures 2.2.10.-1 and 2.2.10.-2,
respectively. For laminar flow, the viscosity (or apparent
viscosity) 11 expressed in pascal-seconds is given by the
following formula:
Figure 2.2.10.-1
M torque in newton-metres acting on the cylinder

surface,
úJ angular velocity in radians per second,
h height of imrnersion in metres of the inner cylinder in M
the liquid medium,
radius in metres of the inner cylinder,
radius in metres of the outer cylinder,
constant of the apparatus, expressed in radians per
cubic metre.
For non-Newtonian liquids it is indispensable to specify the
shear stress (e) or the shear rate (y) at which the viscosity is
measured. Under narrow gap conditions (conditions satisfied h
in absolute viscometers), there is a proportional relationship
between M and e and also between úJ and y:
T=AM ¡=Bw
Figure 2.2.10.-2
V-A246 Appendix V H 2014
Gone-plate viscomelers (absolute viscomelers)

In the cone-plate viscometer, the liquid is introduced into the
gap between a fiat disc and a cone forming a define angle.
Viscosity measurement can be performed by rotating the
cone or the fiat disc, as shown in Figures 2.2.10.-3 and
2.2.10.-4, respectively. For laminar fiow, the viscosity (or
apparent viscosity) 11 expressed in pascal-seconds is given by
the following formula: /\
=kM
w
M torque in Newton-metres acting on the fiat disc or

cone surface,
ro angular velocity in radians per second,
a angle in radians between the fiat disc and the cone,
R radius in metres of the cone,
k constant of the apparatus, expressed in radians per
cubic metre.
Figure 2.2.10.-5
Constants A, B of the apparatus (se e under concentric
cylinder viscometers).
Figure 2.2.10.-3
Figure 2.2.10.-6
In a general way, the constant k of the apparatus may be

M
determined at various speeds of rotation using a certified
viscometer calibration liquido The viscosity 11 then
corresponds to the formula:
M
r¡ = k-
w
Method
Measure the viscosity (or apparent viscosity) according to the
instructions for the operation of the rotating visco meter.
The temperature for measuring the viscosity is indicated in
Figure 2.2.10.-4 the monograph. For non-Newtonian systems, the monograph
indicates the type of viscometer to be used and if absolute
Spindle viscometers (relative viscometers) viscometers are used the angular velocity or the shear rate at
In the spindle viscometer, the viscosity is determined by which the measurement is made. If it is impossible to obtain
rotating a spindle (for example, cylinder- or disc-shaped, as the indicated shear rate exactly, use a shear rate slightly
shown in Figures 2.2.10.-~ and 2.2.10.-6, respectively) higher and a shear rate slightly lower and interpola te .
immersed in the liquido Relative values of viscosity (or With relative viscometers the shear rate is not the same
apparent viscosity) can be directly ca1culated using throughout the sample and therefore it cannot be defined.
conversion factórs from the scale reading at a given Under these conditions, the viscosity of non-Newtonian
rotational speed. liquids determined from the previous formula has a relative
2014 Appendix V J V-A247
character, which depends on the type of spindle and the

angular ve10city as well as the dimensions of the sample
J. Circular Dichroism
container (0 = minimum 80 mm) and the depth of (Ph. Eur. method 2.2.41)
immersion of the spindle. The values obtained are The difference in absorbance of optically active substances
comparable only if the method is carried out under within an absorption band for left and right circularly
experimental conditions that are rigorously the same. polarised light is referred to as circular dichroism.
Direct measurement gives a mean algebraic value:
Method IV (Falling hall viscometer method)
The determination of dynamic viscosity of Newtonian liquids
using a suitable falling ball viscometer is performed at
M circular dichroic absorbance,
20 ± 0.1 oC, unless otherwise prescribed in the monograph.
AL absorbance of left circularly polarised light,
The time required for a test ball to fall in the liquid to be
AR absorbance of right circularly polarised light.
examined from one ring mark to the other is determined.
If no stricter limit is defined for the equipment used the Circular dichroism is calculated using the equation:
result is valid only if 2 consecutive mea sures do not differ by
more than 1.5 per cent. .6. A
.6.é =: éL - éR = - -
Apparatus The falling ball visco meter consists of: a glass ex l
tube enc10sed in a mantle, which allow precise control of
temperature; six balls made of glass, nickel-iron or steel with ll.8 molar circular dichroism or molar differential dichroic
different densities and diameters. The tube is fixed in such a absorptivity expressed in litre·mole-1cm- 1
way that the axis is inc1ined by 10 ± 10 with regard to the eL molar absorptivity (2.2.25) ofleft circularly polarised
vertical. The tube has 2 ring marks which define the distance light,
the ball has to rollo Commercially available apparatus is eR molar absorptivity of right circularly polarised light,
supplied with tables giving the constants, the density of the e concentration of the test solution in mole·litre - 1,
balls and the suitability of the different balls for the expected optical path of the cell in centimetres.
range of viscosity.
The following units may also be used to characterise circular
Method Fill the c1ean, dry tube of the viscometer, dichroism:
previously brought to 20 ± 0.1 oC, with the liquid to be
Dissymmetry factor:
examined, avoiding bubbles. Add the ball suitable for the
range of viscosity of the liquid so as to obtain a falling time
.6.é
not less than 30 s. Close the tube and maintain the solution g=-
at 20 ± 0.1 oC for at least 15 mino Let the ball run through é
the liquid between the 2 ring marks once without

measurement. Let it run again and measure with a stop- e = molar absorptivity (2.2.25).
watch, to the nearest one-fifth of a second, the time required Molar ellipticity
for the ball to roll from the upper to the lower ring mark. Certain types of instruments display directly the value of
Repeat the test run at least 3 times . ellipticity 0, expressed in degrees. When such instruments
Calculate the dynamic viscosity 11 in millipascal seconds are used, the molar ellipticity [0] may be calculated using
using the formula: the following equation:
exM
[e] = -e-x-""Z-x-l-""O
k constant, expressed in millimeter squared per second
squared,
[0] molar ellipticity, expressed in
PI density of the ball used, expressed in grams per cubic
degrees ·cm2 ·decimole- 1,
centimetre,
pz density of the liquid to be examined, expressed in
o value of ellipticity given by the instrument,
M relative molecular mass of the substance to be
grams per cubic centimetre, obtained by multiplying
examined,
its relative density d~g by 0.9982,
e concentration of the solution to be examined in
falling time of the ball, in seconds.
g/mL,
Additional points lor monographs 01 the British optical path of the cell in centimetres.
Pharmacopoeia Molar ellipticity is also related to molar circular dichroism by
The apparatus and methods described in Methods 1 and II the following equation:
are in agreement in all essentials with International Standards
ISO 3104-1994 and 3105-1994 (Methods for the 4500
determination of the viscosity of liquids). [e] = 2.303.6.é-- 'Ir
R; 3300.6.é
Molar ellipticity is often used in the analysis of proteins and

nuc1eic acids. In this case, molar concentration is expressed
in terms of monomeric residue, calculated using the
expression:
molecular mass
number of amino acids
V-A248 Appendix V K 2014
M5
M2
M3
........=---===--==--:-="---'-'===--'-g} P2
Figure 2.2.41.-1. - Optical scheme of a dichrograph
The mean relative molecular mass of the monomeric residue

is 100 to 120 (generally 115) for proteins and about 330 for
K. Relationship Between Reaction of
nucleic acids (as the sodium salt). Solution, Approximate pH and Colour of
Apparatus The light source (S) is a xenon lamp (Figure Certain Indicators
2.2.4l.-1); the light passes through a double monochromator
(M) equipped with quartz prisms (PI, P2). (Ph. Eur. method 2.2.4)
The linear beam from the first monochromator is split into To 10 mL of the solution to be examined, add 0.1 mL of the
2 components polarised at right angles in the second indicator solution, unless otherwise prescribed in
monochromator. The exit slit of the monochromator Table 2.2.4.-l.
eliminates the extraordinary beam. Table 2.2.4.-1
The polarised and monochromatic light passes through a Reaction pH Indicator Colour
birefringent modulator (er): the resuIt is altemating circularly >8
Alkaline Utmus paper red R Blue
polarised light.
Thymol blue Grey or violet-blue
The beam then passes through the sample to be examined solution R (0.05 rnL)
(C) and reaches a photomultiplier (PM) followed by an Slightly alkaline 8.0 - 10.0 Phenolphthalein Colourless or pink
amplifier circuit whlch produces 2 electrical signals: one is a solution R (0.05 rnL)
direct current Ve and the other is an altemating current at Thymol blue Grey
solution R (0.05 rnL)
the modulation frequency V ae characteristic of the sample to
Strongly alkaline >10 Phenolphthalein Red
be examined. The phase gives the sign of the circular paper R
dichroism. The ratio VaJVc is proportional to the differential Thymol blue Violet-blue
absorption M whlch created the signa!. The region of solution R (0.05 rnL)
wavelengths normally covered by a dichrograph is 170 nm to Neutral 6_0 - 8.0 Methyl red solution R Yellow
800 nm. Phenol red solution R
CALIBRATION OF THE APPARATUS (0.05 mL)
Neutral to rnethyl 4.5 - 6.0 Methyl red solution R . Orange-red
Accuracy of absorbance scale Dissolve 10.0 mg of red
isoandrosterone R in dioxan R and dilute to 10.0 mL with the Neutral to < 8.0 Phenolphthalein Colourless; pink or red
same solvento Record the circular dichroism spectrum of the phenolphtalein solution R (0.05 rnL) after adding 0.05 rnL
solution between 280 nm and 360 nm. Measured at the of 0.1 M base
maximum at 304 nm, 68 is + 3.3. Acid <6 Methyl red solution R Orange or red
The solution of (1S)-(+)-lO-camphorsuifonic acid R may also Bromothymol blue Yellow

solution Rl
be used.
Slightlyacid 4.0 - 6.0 Methyl red solution R Orange
Linearity ofmodulation Dissolve 10.0 mg of (1S)-(+)-
JO-camphorsuifonic acid R in water R and dilute to 10.0 mL Bromocresol green Green or blue
solution R
with the saine solvento Determine the exact concentration of Strongly acid <4 Congo red paper R Green or blue
camphorsulfonic acid in the solution by ultraviolet
spectrophotometry (2.2.25), taking the specific absorbance to
be 1.49 at 285 nm.
Record the circular dichroism spectrum between 185 nm and
340 nm. Measured at the maximum at 290.5 nm, 68 is + L. Determination of pH Values
2.2 to + 2.5 . Measured at the maximum at 192.5 nm, 68 is (Ph_ Eur. method 2.2.3)
- 4.3 to - 5. The pH is a number whlch represents conventionally the
(1S)-(+) - or antipodal (1R)-(-)-ammonium hydrogen ion concentration of an aqueous solution.
1O-camphorsuifonate R can also be used. For practical purposes, its definition is an experimental one.
The pH of a solution to be examined is related to that of a
reference solution (pHs) by the following equation:
E-Es
pH = pHs - --k-
2014 Appendix V L V -A249
in which E is the potential, expressed in volts, of the cell All solutions to be examined and the reference buffer
containing the solution to be examined and Es is the solutions must be prepared using caI'bon dioxide-free water R .
potential, expressed in volts, of the cell containing the
solution of known pH (pH s), k is the change in potential per Preparatíon of reference buffer solutions
unit change in pH expressed in volts, and ca1culated from the Potassíum Tetraoxalate 0.05 M Dissolve 12.61 g of
Nernst equation. C 4H 3KO s,2H 2 0 in carbon dioxide-free water R and dilute to
1000.0 mL with the same solvento
Table 2.2.3.-1. - Values of k al differenl lemperalures
Temperature ('C) k (V)
Potassium Hydrogen Tartrate saturated at 25 oC
Shake an excess of C 4H sK0 6 vigorously with caI'bon dioxide-
15 0.0572 free water R at 25 oc. Filter or decanto Prepare irnrnediately
20 0.0582 before use.
25 0.0592 Potassium Dhydrogen Citrate 0.05 M Dissolve 11.41 g
of C 6 H 7K0 7 in caI'bon dioxide-free water R and dilute to
30 0.0601
1000.0 mL with the same solvento Prepare immediately
35 0.0611 before use.
Potassium Hydrogen Phthalate 0.05 M Dissolve
The potentiometric determination of pH is made by 10.13 g of C sH sK0 4 , previously dried for 1 h at
measuring the potential difference between 2 appropriate 110 ± 2 oC, in carbon dioxide-free water R and dilute to
electro des immersed in the solution to be examined: 1 of 1000.0 mL with the same solvento
these electrodes is sensitive to hydrogen ions (usuallya glass Potassium Dihydrogen Phosphate 0.025 M + Disodium
electrode) and the other is the reference electrode (for Hydrogen Phosphate 0.025 M Dissolve 3.3 9 g of
example, a saturated calomel electrode). KH 2P0 4 and 3.53 g ofNa2HP04, both previously dried for
Apparatus The measuring apparatus is a voltrneter with 2 h at 120 ± 2 oC, in carbon dioxide-free water R and dilute
an input resistance at least 100 times that of the electrodes to 1000.0 mL with the same solvento
used. It is normally graduated in pH units and has a Potassium Dihydrogen Phosphate 0.0087 M +
sensitivity such that discrimination of at least 0.05 pH unit Disodium Hydrogen Phosphate 0.0303 M Dissolve
or at least 0.003 V may be achieved. 1.18 g of KH 2P0 4 and 4.30 g of Na2HP04, both previously
Method Unless otherwise prescribed in the monograph, all dried for 2 h at 120 ± 2 oC, in carbon dioxide-free water R
measurements are made at the same temperature and dilute to 1000.0 mL with the same solvent.
(20-25 oC). Table 2.2 .3.-2 shows the variation of pH with Disodium Tetraborate 0.01 M Dissolve 3.80 g of
respect to temperature of a number of reference buffer Na2B40 7,lOH20 in carbon dioxide-free water R and dilute to
solutions used for calibration. For the temperature 1000.0 mL with the same solvento Store protected from
correction, when necessary, follow the manufacturer's atrnospheric carbon dioxide.
instructions. The apparatus is calibrated with the buffer
Sodium Carbonate 0.025 M + Sodium Hydrogen
solution of potassium hydrogen phthalate (primary standard)
Carbonate 0.025 M Dissolve 2.64 g of Na2CO} and
and 1 other buffer solution of different pH (preferably one
2.09 g of NaHC()3 in carbon dioxide1ree water R and dilute
shown in Table 2.2.3.-2). The pH of a third buffer solution
to 1000.0 mL with the same solvento Store protected from
of intermediate pH read off on the scale must not differ by
atrnospheric carbon dioxide.
more than 0.05 pH unit from the value corresponding to this
solution. Immerse the electrodes in the solution to be Calcium Hydroxide, Saturated at 25 oC Shake an
examined and take the reading in the same conditions as for excess of calcium hydroxide R with carbon dioxide-free water R
and decant at 25 oc. Store protected from atrnospheric
the buffer solutions.
carbon dioxide .
When the apparatus is in frequent use, checks must be
carried out regularly. If not, such checks should be carried
out before each measurement.
Table 2.2.3.-2. - pH of reference buffer solutions al various temperalures

Temperature Potassium Potassium Potassium Potassium Potassium Potassium Disodium Sodlum Calcium
('C) tetraoxalate hydrogen dihydrogen hydrogen dihydrogen dlhydrogen tetraborate carbonate hydroxide,
0.05 M tartrate citrate phthalate phosphate phosphate 0.01 M 0.025 M saturated at
saturated at 0.05 M 0.05 M 0.025 M 0.0087 M + 25'C
25 'C + + sodium
disodium disodium bicarbonate
hydrogen hydrogen 0.025 M
phosphate phosphate
0.025 M 0.0303 M
C,H,KO.,2H,O C,H5KO, C,H,KO, C.H,KO, KH,PO,+ KH,PO,+ Na,B,O" Na,CO,+ Ca(OH),
Na,HPO, Na,HPO, IOH,O NaHCO,
15 1.67 3.80 4.00 6.90 7.45 9.28 10.12 12.81
20 1.68 3.79 4.00 6.88 7.43 9.23 10.06 12.63
25 1.68 3.56 3.78 4.01 6.87 7.41 9.18 10.01 12.45
30 1.68 3.55 3.77 4.02 6.85 7.40 9.14 9.97 12.29
35 1.69 3.55 3.76 4.02 6.84 7.39 9.10 9.93 12.13
f:lpH (l)
+ 0.001 -0.0014 -0.0022 + 0.0012 - 0.0028 - 0.0028 -0.0082 -0.0096 -0.034
t:.t
(1) pH variation per degree Celsius.
V-A250 Appendix V M 2014
Storage The technique is used to determine the difference in the fiow

Store buffer solutions in suitable chemicaIly resistant, tight of heat (wi!h reference to the temperature) evolved or
containers, such as type 1 glass bottIes or plastic containers absorbed by !he test sample compared wi!h the reference
suitable for aqueous solutions. ceIl, as a function of the temperature. Two types of DSC
apparatuses are available, !hose using power compensation to
Additional points lor monographs 01 the British maintain a nuIl temperature difference between sample and
Pharmacopoeia reference and !hose !hat apply a constant rate of heating and
[Suitable glass electrodes and pH meters of bo!h !he detect temperature differential as a difference in heat fiow
analogue and digital type are described in British Standards between sample and reference.
2586: 1979 and 3145 : 1978.] Apparatus The apparatus for the power compensation
DSC consists of a fumace containing a sample holder with a
reference ceIl and a test cel!. The apparatus for the heat fiow
M. Thermal Analysis DSC consists of a fumace containing a single ceIl with a
sample holder for !he reference crucible and !he test crucible.
A temperature-programming device, !hermal detectores) and
Thermal analysis is a group of techniques in which !he
a recording system which can be connected to a computer
variation of a physical property of a substance is measured as
are attached. The measurements are carried out under a
a function of temperature. The most commonly used
controlIed atrnosphere.
techniques are !hose which measure changes of mass or
changes in energy of a sample of a substance.
A
Thermogravimetry
Thermogravimetry is a technique in which !he mass of a
sample of a substance is recorded as a function of
temperature according to a controIled temperature
programme.
Apparatus The essential components of a !hermobalance
are a device for heating or cooling !he substance according to
a given temperature program, a sample holder in a Endotherme
controIled atrnosphere, an electrobalance and a recorder. ~ - - - - - - - - - 1- 'C- ________ ~ Temperature
In sorne cases !he instrument may be coupled to a device
permitting !he analysis of volatile products.
Temperature verification Check !he temperature scale Figure 2.2.34.-1. - Thermagram
using a suitable material according to !he manufacturer's
instructions. Calibration of the apparatus Calibrate the apparatus for
Verification ofthe electrobalance Place a suitable temperature and enthalpy change, using indium of high
quantity of a suitable certified reference material (for purity or any" other suitable certified material, according to
example, calcium axalate manahydrate CRS) in !he sample !he manufacturer's instructions. A combination of 2 metals,
holder and record !he mass. Set !he heating rate according to e.g. indium and zinc may be used to control linearity.
rhe manufacturer's instructions and start !he temperature Operating procedure Weigh in a suitable crucible an
increase. Record !he !hermogravimetric curve as a graph with appropriate quantity of!he substance to be examined; place
temperature, or time, on the abscissa, increasing from left to it in !he sample holder. Set !he initial and final temperatures,
right, and mass on the ordinate, increasing upwards. Stop and !he heating rate according to !he operating conditions
!he temperature increase at about 230 oC. Measure !he prescribed in the monograph.
difference on !he graph between !he initial and final
Begin !he analysis and record the differential !hermal analysis
mass-temperature plateaux, or mas s-time plateaux, which
curve, wi!h !he temperature or time on the abscissa (values
corresponds to !he loss of mass. The declared loss of mass
increasing from left to right) and !he energy change on the
for the certified reference material is stated on the labe!'
ordinate (specify whether the change is endo!hermic or
Method Apply!he same procedure to !he substance to be exothermic).
examined, using !he conditions prescribed in the monograph.
The temperature at which !he phenomenon occurs (the onset
Calculate !he loss of mass of the substance to be examined
temperature) corresponds to !he intersection (A) of!he
from !he difference measured in !he graph obtained. Express
extension of the baseline with the tangent at !he point of
!he loss of mass as per cent 11 mlm.
greatest slope (infiexion point) of!he curve (se e Figure
If !he apparatus is in frequent use, carry out temperature 2.2.34.-1). The end of!he thermal phenomenon is indicated
verification and calibration regularly. O!herwise, carry out by !he peak of the curve.
such checks before each measurement.
The enthalpy of !he phenomenon is proportional to !he are a
Since !he test a®osphere is critical, !he foIlowing parameters under !he curve limited by !he baseline; !he proportionality
are noted for each measurement: pressure or fiow rate, factor is determined from the measurement of !he heat of
composition of!he gas. fusion of a known substance (e.g., indium) under the same
Differential scanning calorimetry operating conditions.
Differential Scanning Calorimetry (DSC) is a technique !hat Each !hermogram may be accompanied by the foIlowing
can be used to demonstrate !he energy phenomena produced data: conditions employed, record of last calibration, sample
during heating (or cooling) of a substance (or a mixture of size and identification (including !hermal history), container,
substances) and to determine !he changes in en!halpy and atrnosphere (identity, fiow rate, pressure), direction and rate
specific heat and !he temperatures at which !hes e occur. of temperature change, instrument and recorder sensitivity.
2014 Appendix V M V -A251
APPLICATIONS X2 mole fraction of the impurity i.e. the number of

Phase changes Detennination of the temperature, heat molecules of the impurity divided by the total
capacity change and enthalpy of phase changes undergone by number of molecules in the liquid phase (or molten
a substance as a function of temperature. phase) at temperature T (expressed in kelvins),
solid - solid transition: allotropy - polymorphism To melting point of the chemically pure substance, in
glass transition kelvins,
desolvation !1H¡ molar heat of fusion of the substance, in joules,
R gas constant for ideal gases, in
amorphous-crystalline joules·kelvin- 1mole- J •
solid - liquid transition: melting
solid - gas transition: sublimation Hence, the deterrnination of purity by DSC is limited to the
liquid - solid transition: freezing detection of impurities fonning a eutectic mixture with the
recrystallisation principal compound and present at a mole fraction of less
liquid - gas transition: evaporation than 2 per cent in the substance to be examined.
This method cannot be applied to:
Changes in chemical composition Measurement of heat
and temperatures of reaction under given experimental - amorphous substances,
conditions, so that, for example, the kinetics of - solvates or polymorphic compounds that are unstable
decomposition or of desolvation can be detennined. within the experimental temperature range,
Application to phase diagrams Establishment of phase - impurities forming solid solutions with the principal
diagrams for solid mixtures. The establishment of a phase substance,
diagram may be an important step in the prefonnulation and - impurities that are insoluble in the liquid phase or in the
optimisation of the freeze-drying process. melt of the principal substance.
Determination 01 purity The measurement of the heat of During the heating of the substance to be examined, the
fusion and the melting point by DSC enables the impurity melts completely at the temperature of the eutectic
impurity content of a substance to be determined from a mixture. Above this temperature, the solid phase contains
single thennal diagram, requiring the use of only a few only the pure substance. As the temperature increases
milligrams of sample with no need for repeated accurate progressively from the temperature of the eutectic mixture to
measurements of the true temperature. the melting point of the pure substance, the mole fraction of
In theory, the melting of an entirely crystalline, pure impurity in the liquid decreases constantly, since the quantity
substance at constant pressure is characterised by a heat of of liquified pure substance increases constantly. For all
fusion !1H¡ in an infinitely narrow range, corresponding to temperatures aboye the eutectic point:
the melting point To. A broadening of this range is a sensitive
indicator of impurities. Hence, samples of the same (2)
substance, whose impurity contents vary by a few tenths of a
per cent, give thennal diagrams that are visually distinct (se e
Figure 2.2.34.-2) . F = molten fraction of the analysed sample,
x; = molefraction of the impurity in the analysed sample.
10lIl 1 oC ~I Temperature When the entire sample has melted, F = 1 and X2 = xi.
.............. ~--;..-- ~
If equation (2) is combined with equation (1), the following
--- ......
./......... 1//
equation is obtained:
............................... '~""{ T = To - x'2RT5 x ..!..

!:J.H¡ F
, 1\
ilH
99.10:··\·\.... \'l, \\ I
The value of the heat of fusion is obtained by integrating the
melting peak.
+
o
"O
e
~ ........: ,
,
~
1\
The melting point To of the pure substance is extrapolated
LlJ 99.35% ~' / \ from the plot of lIF versus the temperature expressed in
kelvins. The slope CI. of the curve, obta\ned after linearisation,
9950% \./
. ~
if necessary, corresponding to RT6 /::,.xJ¡ f allows xi to be
evaluated.
99.80%
The fraction xi, multiplied by 100 gives the mole fraction in
per cent for the total eutectic impurities.
Figure 2.2.34.·2. - Thermal diagrams accordirzg to purity
Thennomicroscopy
The detennination of the molar purity by DSC is based on Phase changes may be visualised by thermomicroscopy, a
the use of a mathematical approximation of the integrated method which enables a sample subjected to a programmed
fonn of the Van't Hoff equation applied to the temperarure change to be examined, in polarised light, under
concentrations (not the activities) in a binary system a microscope.
[In (1 -X2) = - X2 and T x To = TJ]: The observations made in thennomicroscopy allow the
nature of the phenomena detected using thennogravimetry
RT5 and differential thennal analysis to be c1early identified.
T=To - - - x X2 (1)
!:J.H¡
V-A252 Appendix V N 2014
Table 2.2.35 .-1. - Re(erence solutions (or osmometer

Apparatus The apparatus consists of a microscope fitted
calib ratio n
with a light polariser, a hot plate, a temperature and heating
Mass in grams oC Real Ideal Molal osmotic Cryoscopic
rate ami/or cooling rate programmer and a recording system sodium chloride R osmolality osmolality coeCficient depression
for the transition temperatures. A video camera and video per kilogram oC (mosmol/kg) (mosmol/kg) (OC)
recorder may be added. water R
3.087 100 105.67 0.9463 0. 186
6.260 200 214.20 0.9337 0.372
9.463 300 323.83 0.9264 0.558

N.Osmolality
12.684 400 434.07 0.9215 0.744
15.916 500 544.66 0.9180 0.930
Osmolality is a practica! means of giving an overall measure
of the contribution of the various solutes present in a solution 19.147 600 655.24 0.9157 1.116
to the osmotic pressure of the solution. 765.86 0.9140 1.302
22.380 700
An acceptable approximation for the osmolality ~m of a given
aqueous solution is given by:
Carry out the same operations with the test sample. Read
directly the osmolality or calculate it from the measured
depression of freezing point. The test is not valid unless the
value found is within 2 values of the calibration scale.
If the solute is not ionised, v = 1; otherwise \l is the total
number of ions already present or formed by solvolysis from
1 molecule of solute.
molality of the solution, that is the number of moles of O. Conductivity
solute per kilogram of solvent;
molal osmotic coefficient which takes account of the
interactions between ions of opposite charge in the The current 1 (in amperes) fiowing in a conductor is directly
solution. It is dependent on the value of m. As the proponional to the applied electromotive force E (in volts)
complexity of solutions increases, !]l becomes difficult and inversely proportional to the resistance R (in ohms) of
to measure. the conductor:
The unit of osmolality is osmole per kilogram (osmol/kg), but 1= ~

R
the submultiple milliosmole per kilogram (mosmol/kg) is
usually used. The conductivity (formerly called specific conductance) of a
solution (K) is, by definition, the reciprocal of resistivity (p).
Unless otherwise prescribed, osmolality is determined by
Resistivity is defined as' the quotient of the electric field and
measurement of the depression of freezing point.
the density of the current. The resistance R (in n ) of a
The following relationship exists between the osmolality and
conductor of cross-section S (in cm 2) and length L (in cm) is
the depression of freezing point L1 T:
given by the expression:
6.T L
~m = 1.86 x 1000 mosmol/ kg R=p -
S
Thus:
Apparatus The apparatus (osmometer) consists of:
L L
- a means of cooling the container used for the R = -1
/í,
x - or /í,
S
= -R1 x -
S
measurement;
- a system for measuring temperature consisting of a resistor LIS corresponds to the ideal cell constant.
sensitive to temperature (thermistor), with an appropriate The unit of conductivity in the International System is the
current or potential-difference measurement device that siemens per metre (S· m - 1). In practice, the electrical
may be graduated in temperature depression or directly in conductivity of a solution is expressed in siemens
osmolality; per centimetre (S·cm- 1) or in microsiemens per centimetre
- a means of mixing the sample is usually included. (f.lS·cm- 1). The unit of resistivity in the International System
is the ohm-metre (n·m). The resistivity of a solution is
Method Prepare reference solutions as described in
generally expressed in ohm-centimetres (n·cm). Unless
Table 2.2.35.-1, as required. Determine the zero of the
otherwise prescribed, the reference temperature for the
apparatus using water R . Calibrate the apparatus using the
expression of conductivity or resistivity is 25 oc.
reference solutions: introduce a suitable volume of sample
into the measurement cell, as indicated by the equipment The apparatus and operating procedure described below are
supplier, and stan the cooling system. Usually, the mixing applicable to laboratory measurement of conductivity greater
device is programmed to operate at a temperature below that than 10 f.lS ·cm - 1 . The measurement ofconductivity ofwater
expected through ctyoscopic depression to prevent is dealt with in the relevant monographs.
supercooling; A suitable device indicates attainment of
Apparatus
equilibrium . Before each measurement, rinse the
measurement cell with the solution to be examined. The apparatus used (conductivity meter or resistivity meter)
mea sures the resistance of the column of liquid between the
electro des of the immersed m easuring device (conductivity
cell) . The apparatus is supplied with alternating current to
avoid the effects of electrode polarisation. It is equipped with
a temperature probe and a temperature compensation device.
2014 Appendix V P V-A253
The conductivity cell contains 2 parallel platinum electrodes monitor the performance of various operations in the
coated with platinum black, each with a surface area S, and preparation of medicines.
separated from the other by a distance L. Both are generally A variety of acceptable methods is available for determining
protected by a glass tube. Other types of cells may also be TOC. Rather than prescribing a given method to be used,
used. this general chapter describes the procedures used to qualify
the chosen method and the interpretation of results in limit
Operating procedure
tests. A standard solution is analysed at suitable intervals,
DETERMINATION OF THE CELL CONSTANT depending on the frequency of measurements; the solutionis
Choose a conductivity cell that is appropriate for the prepared with a substance that is expected to be easily
properties and conductivity of the solution to be examined. oxidisable (for example, sucrose) at a concentration adjusted
The higher the expected conductivity, the higher the cell to give an instrument response corresponding to the TOC
constant that must be chosen (low p). Commonly used limit to be measured. The suitability of the system is
conductivity cells have cell constants of the order of determined by analysis of a solution prepared with a
0.1 cm- \ 1 cm- l and 10 cm-l. Use a certified reference substance expected to be oxidisable with difficulty (for
material, for example a solution of potassium chloride, that is example, l,4-benzoquinone).
appropriate for the measurement. The conductivity value of
The various types of apparatus used to measure TOC in
the certified reference material, should be near the expected
water for pharmaceutical use have in common the objective
conductivity value of the solution to be examined. Other
of completely oxidising the organic molecules in the sample
certified reference materials may be used especially for cells
water to produce carbon dioxide followed by measurement of
having a constant ofO.1 cm-l . Rinse the cell several times
the amount of carbon dioxide produced, the result being
with distilled water R and at least twice with the certified
used to calculate the carbon concentration in the water.
reference material used for the determination of the cell
constant of the conductivity cell. Measure the resistance of The apparatus used must discriminate between organic and
the conductivity cell using the certified reference material at inorganic carbon, the latter being present as carbonate.
25 ± 1 °C. The cell constant Keell (in cm- 1 ) depends on the The discrimination may be effected either by measuring the
geometry of the conductivity cell and is given by the inorganic carbon and subtracting it from the total carbon, or
expression: by purging inorganic carbon from the sample before
oxidisation. Purging may also entra in organic molecules, but
Kcell = RCRM X "'CRM such purgeable organic carbon is present in negligible
RCRM measured resistance, expressed in mega-ohms quantities in water for pharmaceutical use.
KCRM conductivity of the certified reference material Apparatus
solution used, expressed in microsiemens
per centimetre Use a calibrated instrument installed either on-line or off-
lineo Verify the system suitability at suitable intervals as
The measured constant Kall of the conductivity cell must be described below. The apparatus must have a limit of
within 5 per cent of the value indicated. detection specified by the manufacturer of 0.05 mg or less of
If the determination of the cell constant is carried out at a carbon per litre.
different temperature than that indicated for the certified TOCwater
reference material, the conductivity value may be calculated
Use highly purified water complying with the following
from the following expression:
specifications:
"'T = "'TCRM x [l+a(T-TCRM)] - conductivity: not greater than 1.0¡.tS·cm- 1 at 25 oC,
KT value of conductivity at the different temperature - total organic carbon: not greater than 0.1 mglL.
KTCRM value of conductivity of the certified reference Depending on the type of apparatus used, the content of
material heavy metal s and copper may be critica!. The manufacturer's
T temperature set for calibration instructions should be followed.
TCRM temperature indicated for the certified reference
material Glassware preparation
temperature coefficient for the conductivity value Use glassware that has been scrupulously c1eaned by a
of the certified reference material; for potassium method that will remove organic matter. Use Toe water for
chloride CL = 0.021. the final rinse of glassware.
DETERMINATION OF THE CONDUCTIVITY OF THE Standard solution
SOLUTION TO BE EXAMINED Dissolve sucrose R, dried at 105 oC for 3 h in TOe water to
After calibrating the apparatus with a certified reference obtain a solution containing 1.19 mg of sucrose per litre
material solution, rinse the conductivity cell several times (0.50 mg of carbon per litre).
with distilled water R and at least twice with the aqueous
Test solution
solution to be examined. Carry out successive measurements
as described in the monograph. Using all due care to avoid contamination, collect water to be
tested in an airtight container leaving minimal head-space.
Examine the water with minimum delay to reduce
contamination from the container and its c1osure.
P. Total Organic Carbon in Water for System suitability solution
Pharmaceutical Use Dissolve 1,4-benzoquinone R in TOe water to obtain a
(Ph. Eur. method 2.2.44) solution having a concentration of 0.75 mg of
Total organic carbon (TOC) determination is an indirect l,4-benzoquinone per litre (0.50 mg of carbon per litre).
measure of organic substances present in water for
pharmaceutical use. TOC determination can also be used to
V-A254 Appendix V Q 2014
TOC water control True density

Use TOC water obtained at the same time as that used to The true density of a substance is the ratio of the mass to the
prepare the standard solution and the system suitability volume of the unit cell, exclusive of all voids that are not a
solution. fundamental part of the molecular packing arrangement. It is
Control solutions an intrinsic property of the specified crystal structure of
substance, and hence should be independent of the method
In addition 10 the TOe water control, prepare suitable blank
of determination. The true density is determined by
solutions or other solutions needed for establishing the
calculation.
baseline or for calibration adjustments following the
m anufacturer's instructions; run the appropriate blanks 10 It is obtained using crystallographic data (volume and
zero the instrumento composition of the unit cell) from, for example, X-ray
diffraction data, either on a single crystal or by refinement of
System suitability the crystalline structure from X-ray powder diffraction data.
Run the following solutions and record the responses: TOe
water (rw); standard solution ,(rs); system suitability solution (rss ). Particle density
Calculate the percentage response efficiency using the The particle density takes into account both the true density
expression: and the intraparticulate porosity (sealed and/or
experimentally non-accessible open pores). Thus, particle
Tss - Tw
x 100 density depends on the value of the volume determined,
Ts - rw which in turn depends on the method of measurement.
The particle density can be determined using one of the 2
The system is suitable if the response efficiency is not less following methods.
than 85 per cent and not more than 115 per cent of the The gas pycnometric density is determined by measuring the
theoretical response. volume occupied by a known mass of powder, which is
Procedure equivalent to the volume of gas displaced by the powder
Run the test solution and record the response (ru). The test using a gas displacement pycnometer (2.9.23) . In gas
solution complies with the test if ru is not greater than rs - rw . pycnometric density measurements, the volume determined
excludes the volume occupied by open pores; however, it
The method can also be applied using on-line
includes the volume occupied by sealed pores or pores
instrumentation that has been adequately calibrated and
inaccessible to the gas. Due 10 the high diffusivity of helium,
shown 10 have acceptable system suitability. The location of
which is the preferred choice of gas, most open pores are
instrumentation must be chosen to ensure that the responses
accessible 10 the gas. Therefore, the gas pycnometric density
are representative of the water used.
of a finely milled powder is generally not very different from
the rrue density. Hence, this density is the best estimate of
the true density of an amorphous or partially crystalline
sample and is therefore widely applicable for processed
Q. Density of Solids pharmaceutical powder samples.
(Ph. Eur. method 2.2.42) The mercury porosimeter density is also called granular density.
The density of solids corresponds to their average mas s per With this method the volume determined includes the
unit volume and typically is expressed in grams per cubic volume occupied by sealed pores or pores inaccessible lO
centimetre (g/cm 3) although the International Unit is the mercury; however, it includes the volume only from open
kilogram per cubic meter (1 g/cm3 = 1000 kg/m 3). pores smaller than sorne size limito This pore-size limit or
Unlike gases and liquids whose density depends only on minimal access diameter depends on the maximal mercury
temperature and pressure, the density of a solid also depends intrusion pressure applied during the measurement, and
on its assembly and therefore varies with the crystal structure under normal operating pressures the mercury does not
and degree of crystallinity. penetrate the finest pores accessible 10 helium. Various
When a solid is amorphous or partially amorphous, its granular densities can be obtained from one sample since, for
density may further depend upon the history of preparation, each applied mercury intrusion pressure, a density can be
treatment and storage. determined that corresponds to the pore-size limit at that
pressure.
Therefore, unlike fiuids, the densities of 2 chemically
equivalent solids may be different, and this difference refiects BuIk and tapped density
a difference in solid-state structure. The density of
constituent particles is an important physical characteristic of The bulk density of a powder includes the contribution of
pharmaceutical powders. interparticulate void volume. Hence, the bulk density
depends on both the density of powder particles and the
The density of a solid particle can assume different values
spatial arrangement of particles in the powder bed.
depending on the method used to measure the volume of the
particle. It is useful to distinguish 3 levels of expression of The bulk density of a powder is often very difficult 10
density: measure with good reproducibility since the slightest
disturbance of the bed may result in a new density. Thus, it
- the true density, which only includes the solid fraction of
is essential in reporting bulk density to specify how the
the material; in case of crystalline material, the true
determination was made.
density is also called crystal density;
The bulk density and the tapped density are determined as
- the particle density, which also includes the volume due 10
mentioned in chapter 2.9.34. Bulk density and tapped densilJ!.
intraparticulate pores;
- the bulk density, which further includes the interparticulate
void volume formed in the powder bed.
2014 Appendix VI V-A255
that escapes just beneath the surface of a mixture of 1 mL of

Appendix VI 0.1 M hydrochloric acid and 0.05 mL of methyl red solution R .
The colour of the indica10r changes 10 yellow. On addition of
1 mL of a freshly prepared 100 giL solution of sodiwn
Qualitative Reactions and Tests cobaltinitrite R a yellow precipitate is formed.
Ammonium Salts and Salts of Volatile Bases
Acetates Dissolve about 20 mg of the substance 10 be examined in
A. Heat the substance to be examined with an equal 2 mL of water R or use 2 mL of the prescribed solution.
quantity of oxalic acid R. Acid vapours wirh the Add 2 mL of dilute sodium hydroxide solution R. On heating,
characteristic odour of acetic acid are Iiberated, showing the solution gives off vapour that can be identified by its
an acid reaction (2.2.4). odour and by its alkaline reaction (2.2.4).
B. Dissolve about 30 mg of the substance to be examined
in 3 mL of water R or use 3 mL of the prescribed Antimony Compounds
solution. Add successively 0.25 mL of lanthanum nitra te Dissolve with gentle heating about 10 mg of the substance to
solution R, 0.1 mL of 0.05 M iodine and 0.05 mL of be examined in a solution of 0.5 g of sodium potassium
dilute ammonia R2. Heat carefully ro boiling. Within a tamate R in 10 mL of water R and allow 10 cool: to 2 mL of
few minutes a blue precipitate is formed or a dark blue this solution, or 10 2 mL of the prescribed solution, add
colour develops. sodium sulfide solution R dropwise; an orange-red precipitate is
formed which dissolves on addition of dilute sodium hydroxide
Acetyl Groups
solution R.
In a test-tube about 180 mm long and 18 mm in external
diameter, place about 15 mg of the substance to be Arsenic Compounds
examined, or the prescribed quantity, and 0.15 mL of Heat 5 mL of the prescribed solution on a water-bath with
phosphoric acid R. Close the tube with a stopper through an equal volume of hypophosphorous reagent R. A brown
which passes a small test-tube about 100 mm long and precipitate is formed.
10 mm in external diameter containing water R to act as a
condenser. On the outside of the smaller tube, hang a drop Barbiturates, Non-nitrogen Substituted
of lanthanum nitrate solution R. Except for substances
Dissolve about 5 mg of the substance to be examined in
hydrolysable only with difficulty, place the apparatus in a
3 mL of methanol R, add 0.1 mL of a solution containing
water-bath for 5 min, then take out the smaller tube. Remove
100 giL of cobalt nitrate R and 100 gIL of calcium chloride R.
the drop and mix ir with 0.05 mL of 0.01 M iodine on a tile.
Mix and add, with shaking, 0.1 mL of dilute sodium hydroxide
Add at the edge 0.05 mL of dilute ammonia R2. After 1 min
solution R. A violet-blue colour and precipitate are formed.
to 2 min, a blue colour develops at the junction of the two
drops; the colour intensifies and persists for a short time. Benzoates
For substances hydrolysable only with difficulty heat the mixture
A. To 1 mL ofthe prescribed solution add 0.5 mL ofjerric
slowly 10 boiling over an open fiame and then proceed as
chloride solution Rl. A dull-yellow precipitate, soluble in
prescribed aboye.
ether R, is formed.
AIkaloids B. Place 0.2 g of the substance to be examined, treated if
Dissolve a few milligrams of the substance to be examined, necessary as prescribed, in a test-tube. Moisten with
or the prescribed quantity, in 5 mL of water R, add dilute 0.2 mL 10 0.3 mL of sulfuric acid R. Gently warm the
hydrochloric acid R until an acid reaction occurs (2.2.4), then bottom of the tube. A white sublimate is deposited on
1 mL of potassium iodobismuthate solutian R. An orange or the inner wall of the tube.
orange-red precipitate is formed immediately. C . Dissolve 0.5 g of the substance to be examined in
10 mL of water R or use 10 mL of the prescribed
Aluminium Salts solution. Add 0.5 mL of hydrochloric acid R.
Dissolve about 15 mg of the substance to be examined in The precipitate obtained, after crystallisation from warm
2 mL of water R or use 2 mL of the prescribed solution. water R and drying in vacuo, has a melting point
Add about 0.5 mL of dilute hydrochloric acid R and about (2.2. 14) of 120 oC ro 124 oc.
0.5 mL of thioacetamide reagent R. No precipitate is formed.
Add dropwise dilute sodium hydroxide solution R. A gelatinous Bicarbonates
white precipitate is formed which dissolves on further A. Introduce into a test tube 0.1 g of the substance being
addition of dilute sodium hydroxide solution R. Gradually add examined suspended in 2 mL of water or use 2 mL of
ammonium chloride solution R. The gelatinous white precipitate the prescribed solution. Add 3 mL of 2M acetic acid,
is re-formed. close the tube immediately using a s10pper fitted with a
glass rube bent at two right angles. The solution or
Amines, Primary Aromatic
suspension effervesces. Heat gently and collect the gas in
Acidify the prescribed solution with dilute hydrochloric acid R 5 mL of a 4.73% w/v solution of barium hydroxide.
and add 0.2 mL of sodium nitrite solurion R. After 1 min to A white precipitate is produced which dissolves on
2 min, add 1 mL of fJ-naphthol solution R. An intense orange addition of an excess of 7M hydrochloric acid.
or red colour and usually a precipitate of the same colour are
B. Treat a solution of the substance being examined with a
produced.
solution of magnesium sulfate; no precipitate is produced
Ammonium Salts (distinction from carbonates). Boil; a white precipitate is
produced.
To the prescribed solution add 0.2 g of magnesium oxide R.
Pass a current of air through the mixture and direct the gas C . A solution libera tes carbon dioxide when boiled.
V-A256 Appendix VI 2014
-----------------------------------------------
Bismuth and Bismuth Compounds C. To 5 mL of 0.4% w/v solution of the substance being
A. To 0.5 g ofthe substance to be examined add 10 mL of examined add 0.2 mL of a 2% w/v solution of
dz7ute hydroehlone acid R or use 10 mL of the prescribed ammonium oxalate. A white precipitate is produced which
solution. Heat to boiling for 1 mino Cool and filter if is only sparingly soluble in 6M aeetie acid but is soluble in
necessary. To 1 mL of the solution obtained add 20 mL hydrochloric acid.
of water R. A white or slightly yellow precipitate is
Carbonates
formed which on addition of 0.05 mL to 0.1 mL of
sodium sulfide solution R tums brown. For monographs from the European Pharmaeopoeia, use test A
only.
B. To about 45 mg of the substance to be examined add
10 mL of dilute nitne aeid R or use 10 mL of the A. Introduce into a test tube 0.1 g of the substance being
prescribed solution. Boil for 1 minoAllow to cool and examined suspended in 2 mL of water or use 2 mL of
filter if necessary. To 5 mL of the solution obtained add the prescribed solution. Add 3 mL of 2M aeetie acid,
2 mL of a 100 gIL solution of thiourea R. A yellowish- close the tube immediately using a stopper fitted with a
orange colour or an orange precipitate is formed. glass tube bent at two right angles. The solution or
Add 4 mL of a 25 giL solution of sodium fiuonde R. suspension effervesces evolving a colourless and
The solution is not decolorised within 30 mino odourless gas. Heat gently and collect the gas in 5 mL of
O.lM banum hydroxide. A white precipitate is produced
Bromides which dissolves on addition of an excess of
7M hydroehlonc aeid.
A. Dissolve in 2 mL of water R a quantity of the substance
to be examined equivalent to about 3 mg of bromide B. Treat a solution of the substance being examined with a
(Br-) or use 2 mL of the prescribed solution. Acidify solution of magnesium sulfate. A white precipitate is
with dilute nime acid R and add 0.4 mL of sz7ver nitrate produced (distinction from bicarbonates) .
solution RJ. Shake and allow to stand. A curdled, pale
Carbonates and Bicarbonates
yellow precipitate is formed. Centrifuge and wash the
precipitate with three quantities, each of 1 mL, of Introduce into a test-tube 0.1 g of the substance to be
water R . Carry out this operation rapidly in subdued examined and suspend in 2 mL of water R or use 2 mL of
light disregarding the fact that the supematant solution the prescribed solution. Add 3 mL of dilute aeetie aeid R.
may not become perfectly clear. Suspend the precipitate Close the tube irnmediately using a stopper fitted with a glass
obtained in 2 mL of water R and add 1.5 mL of tube bent twice at right angles. The solution or the
ammonia R. The precipitate dissolves with difficulty. suspension becomes effervescent and gives off a colourless
B. Introduce into a small test-tube a quantity of the and odourless gas. Heat gently and collect the gas in 5 mL of
substance to be examined equivalent to about 5 mg of banum hydroxide solution R. A white precipitate is formed that
bromide (Brl or the prescribed quantity. Add 0.25 mL dissolves on addition of an excess of hydroehlonc acid RJ.
of water R, about 75 mg of lead dioxide R, 0.25 mL of Ch10rides
aeetie acid R and shake gently. Dry the inside of the
upper part of the test-tube with a piece of filter paper A. Dissolve in 2 mL of water R a quantity of the substance
and allow to stand for 5 mino Prepare a strip of suitable to be examined equivalent to about 2 mg of chloride
filter paper of appropriate size. Impregnate it by (Cn or use 2 mL of the prescribed solution. Acidify
capillarity, by dipping the tip in a drop of decolonsed with dilute nitnc acid R and add 0.4 mL of silver nitrate
fuehsin solution R and introduce the impregnated part solution RJ. Shake and allow to stand. A curdled, white
irnmediately into the tube. Starting from the tip, a violet precipitate is formed. Centrifuge and wash the
colour appears within lOs that is clearly distinguishable precipitate with three quantities, each of 1 mL, of
from the red colour of fuchsin, which may be visible on water R. Carry out this operation rapidly in subdued
a small area at the top of the impregnated part of the light, disregarding the fact that the supematant solution
paper strip. may not become perfectly clear. Suspend the precipitate
in 2 mL of water R and add 1.5 mL of ammonia R.
CaIcium and Calcium SaIts The precipitate dissolves easily with the possible
exception of a few large particles which dissolve slowly.
For monographs from the European Pharmacopoeia, use tests A
and B only. B. Introduce into a test-tube a quantity of the substance to
be examined equivalent to about 15 mg of chloride (Cn
A. To 0.2 mL of a neutral solution containing a quantity of
or the prescribed quantity. Add 0.2 g of potassium
the substance to be examined equivalent to about
dichromate R and 1 mL of sulfunc acid R. Place a filter-
0.2 mg of ca1cium (Ca 2 +) per millilitre or to 0.2 mL of
paper strip impregnated with 0.1 mL of diphenylcarbazide
the prescribed solution add 0.5 mL of a 2 gIL solution
solution R over the opening of the test-tube. The paper
of g/yoxal-hydroxyanil R in ethanol (96 per eent) R,
tums violet-red. The impregnated paper must not come
0.2 mL of dilute sodium hydroxide solution R and 0.2 mL
into contact with the potassium dichromate.
of sodium carbonate so/ution R. Shake with 1 mL to 2 mL
of ehloroform R and add 1 mL to 2 mL of water R. Citrates
The chloroform layer is coloured red.
For monographs from the European Pharmacopoeia, use test A
B. Dissolve about 20 mg of the substance to be examined only.
or the prescribed quantity in 5 mL of aeetie acid R.
A. Dissolve in 5 mL of water R a quantity of the substance
Actd 0.5 mL of potassium ferrocyanide so/ution R.
to be examined equivalent to about 50 mg of citric acid
The solution remains clear. Add about 50 mg of
or use 5 mL of the prescribed solution. Add 0.5 mL of
ammonium eh/onde R . A white, crystalJine precipitate is
su/fun·c acid R and 1 mL of potassium permanganate
formed. -
solution R. Warm until the colour ofthe permanganate is
discharged. Add 0.5 mL of a 100 gIL solution of sodium
2014 Appendix VI V-A257
nitroprusside R in dilute sulfuric acid R and 4 g of sulfamic Lactates

acid R . Make alkaline with concentrated ammonia R, Dissolve a quantity of the substance to be examined
added dropwise until all the sulfamic acid has dissolved. equivalent to about 5 mg of lactic acid in 5 mL of water R or
Addition of an excess of concentrated ammonia R use 5 mL of the prescribed solution. Add 1 mL of bromine
produces a violet colour, tuming to violet-blue. water R and 0.5 mL of dilute sulfuric acid R. Heat on a water-
B. To a neutral solution of the substance being examined bath until the colour is discharged, stirring occasionally with
add a solution of calcium chloridej no precipitate is a glass rod. Add 4 g of ammonium sulfate R and mix.
produced. Boil the solutionj a white precipitate is Add dropwise and without mixing 0.2 mL of a 100 gIL
produced which is soluble in 6M acetic acid. solution of sodium nitroprusside R in dilute sulfurie acid R. Still
without mixing add 1 mL of coneentrated ammonia R. Allow
Esters ro stand for 30 mino A dark green ring appears at the
To about 30 mg of the substance to be examined or the junction of the two liquids.
prescribed quantity add 0.5 mL of a 70 gIL solution of
hydroxylamine hydrochloride R in methanol R and 0.5 mL of a Lead and Lead Compounds
100 gIL solution of potassium hydroxide R in ethanol A. Dissolve 0.1 g of the substance ro be examined in 1 mL
(96 per cent) R. Heat to boiling, cool, acidify with dilute of aeetie aeid R or use 1 mL of the prescribed solution.
hydrochloric acid R and add 0.2 mL ofjerric chloride Add 2 mL of potassium ehromate solution R. A yellow
solution Rl diluted ten times. A bluish-red or red colour is precipitate is formed that dissolves on addition of 2 mL
produced. of strong sodium hydroxide solution R.
B. Dissolve 50 mg of the substance ro be examined in
Iodides 1 mL of aeetie acid R or use 1 mL of the prescribed
A. Dissolve a quantity of the substance to be examined solution. Add 10 mL of water R and 0.2 mL of potassium
equivalent to about 4 mg of iodide (n in 2 mL of iodide solution R. A yellow precipitate is formed. Heat ro
water R or use 2 mL of the prescribed solution. Acidify boiling for 1 min to 2 mino The precipita te dissolves.
with dilute nitric acid R and add 0.4 mL of silver nitrate Allow to cool. The precipitate is re-formed as glistening,
solution Rl . Shake and allow to stand . A curdled, pale- yellow plates.
yellow precipitate is formed. Centrifuge and wash with
three quantities, each of 1 mL, of water R. Carry out this Lignin
operation rapidly in subdued light disregarding the fact A. Lignin cell walls are coloured bright red by soaking them
that the supematant solution may not become perfectly in a 1% w/v solution of phloroglueinol in ethanol (90%)
c1ear. Suspend the precipitáte in 2 mL of water R and and adding 0.1 to 0.2 mi of hydroehlorie acid.
add 1.5 mL of ammonia R. The precipitate do es not B. Lignified tissues are coloured yellow by aniline
dissolve. hydroehloride solution.
B. To 0.2 mL of a solution ofthe substance ro be
examined containing about 5 mg of iodide (n per Magnesium and Magnesium Salts
millilitre, or to 0.2 mL of the prescribed solution, add For monographs jrom the European Pharmaeopoeia, use test A
0.5 mL of dilute sulfuric acid R, 0.1 mL of potassium only.
dichromate solution R, 2 mL of water R and 2 mL of A. Dissolve about 15 mg of the substance ro be examined
chlorojorm R. Shake for a few seconds and allow to in 2 mL of water R or use 2 mL of the prescribed
stand. The chloroform layer is coloured violet or solution. Add 1 mL of dilute ammonia Rl . A white
violet-red. precipitate is formed that dissolves on addition of 1 mL
of ammonium eh/oride solution R. Add 1 mL of disodium
Iron and Iron Salts hydrogen phosphate solution R. A white crystalline
A. Dissolve a quantity of the substance to be examined precipitate is formed.
equivalent ro about 10 mg of iron (Fé+) in 1 mL of B. To 0.5 mL of a neutral or slightly acidic solution of the
water R or use 1 mL of the prescribed solution. substance being examined add 0.2 mL of a 0.1 % w/v
Add 1 mL of potassium jerricyanide solution R . A blue solution of titan yellow and 0.5 mL of O.IM sodium
precipitate is formed that does not dissolve on addition hydroxide. A bright red turbidity is produced which
of 5 mL of dilute hydrochloric acid R. gradually settles to give a bright red precipitate.
B. Dissolve a quantity of the substance to be examined
equivalent ro about 1 mg of iron (Fe 3 +) in 30 mL of Mercury and Mercury Compounds
water R. To 3 mL of this solution or ro 3 mL of the A. Place about 0.1 mL of a solution of the substance ro be
prescribed solution, add 1 mL of dilute hydrochloric examined on well-scraped copper foil. A dark-grey stain
acid R and 1 mL of potassium thiocyanate solution R. that becomes shiny on rubbing is formed. Dry the foil
The solution is coloured red. Take two portions, each of and heat in a test-tube. The spot disappears.
1 mL, of the mixture. To one portion add 5 mL of B. To the prescribed solution add dilute sodium hydroxide
isoamyl alcohol R or 5 mL of ether R . Shake and allow ro solution R until strongly alkaline (2.2.4). A dense yellow
stand. The organic layer is coloured pink. To the other precipitate is formed (mercuric salts).
portion add 2 mL of mereurie ehloride solution R. The red
colour disappears. Nitrates
C. Dissolve a quantity of the substance ro be examined To a mixture of 0.1 mL of nitrobenzene R and 0.2 mL of
equivalent ro not les s than 1 mg of iron (Fe 3 +) in 1 mL sulfurie aeid R , add a quantity of the powdered substance
of water R or use 1 mL of the prescribed solution. equivalent to about 1 mg of nitrate (N0 3 ) or the prescribed
Add 1 mL of potassium jerroeyanide solution R. A blue quantity. Allow to stand for 5 mino Cool in iced water and
precipitate is formed that does not dissolve on addition add slowly and with mixing 5 mL of water R, then 5 mL of
of 5 mL of dilute hydrochlorie acid R. strong sodium hydroxide solution R. Add 5 mL of aeetone R .
V-A258 Appendix VI 2014
Shake and allow to stand. The upper layer is coloured deep inside of the test-tube with a glass rod. A dense white
violet. precipitate is formed.
B. Dissolve a quantity of the substance to be examined
Odour
equivalent to about 2 mg of sodium (Na +) in 0.5 mL of
(Ph. Eur. method 2.3.4) water R or use 0.5 mL of the prescribed solution.
On a watch-glass 6 cm to 8 cm in diameter, spread in a thin Add 1.5 mL of methoxyphenylacetic reagent R and cool in
layer 0.5 g to 2.0 g of the substance to be examined. After ice-water for 30 min oA voluminous, white, crystaIline
15 min, determine the odour or verify the absence of odour. precipitate is formed. Place in water at 20 oC and stir for
Phosphates (Orthophosphates) 5 mino The precipitate do es not disappear. Add 1 mL of
dilute ammonia R1. The precipitate dissolves completely.
A. To 5 mL of the prescribed solution, neutralised if Add 1 mL of ammonium carbonate solution R .
necessary, add 5 mL of silver nitrate solution R1. A yeIlow No precipitate is formed.
precipitate is formed whose colour is not changed by
boiling and which dissolves on addition of ammonia R . Sulfates
B. Mix 1 mL of the prescribed solution with 2 mL of A. Dissolve about 45 mg of the substance ro be examined
molybdovanadic reagent R . A yeIlow colour develops. in 5 mL of water R or use 5 mL of the prescribed
Potassium and Potassium Salts solution. Add 1 mL of dilute hydrochloric acid R and
1 mL of barium chloride solution R1. A white precipitate is
A. Dissolve 0.1 g of the substance to be examined in 2 mL formed.
of water R or use 2 mL of the prescribed solution.
B. To the suspension obtained during reaction (a), add
Add 1 mL of sodium carbonate solution R and heat.
0.1 mL of 0.05 M iodine. The suspension remains yeIlow
No precipitate is formed. Add to the hot solution
(distinction from sulfites and dithionites), but is
0.05 mL of sodium sulfide solution R. No precipitate is
decolorised by adding dropwise stannous chloride
formed. Cool in iced water and add 2 mL of a 150 gIL
solution R (distinction from iodates). Boil the mixture.
solution of tartaric acid R. AlIow to stand. A white
No coloured precipitate is formed (distinction from
crystaIline precipitate is formed.
selenates and tungstates).
B. Dissolve about 40 mg of the substance to be examined
in 1 mL of water R or use 1 mL of the prescribed Tartrates
solution. Add 1 mL of dilute acetic acid R and 1 mL of a
A. Dissolve about 15 mg of the substance ro be examined
freshly prepared 100 gIL solution of sodium
in 5 mL of water R or use 5 mL of the prescribed
cobaltinitrite R . A yellow or orange-yeIlow precipitate is
solution. Add 0 .05 mL of a 10 gIL solution ofjerrous
formed irnmediately.
sulfate R and 0.05 mL of dilute hydrogen peroxide
Salicylates solution R. A transient yeIlow colour is produced. After
A. To 1 mL of the prescribed solution add 0.5 mL ofjerric the colour has disappeared add dilute sodium hydroxide
chloride solution R1. A violet colour is produced that solution R dropwise. A violet or purple colour is
persists after the addition of 0.1 mL of acetic acid R . produced.
B. Dissolve 0.5 g of the substance to be examined in B. To 0.1 mL of a solution of the substance to be
10 mL of water R or use 10 mL of the prescribed examined containing the equivalent of about 15 mg of
solution. Add 0.5 mL of hydrochloric acid R. tartaric acid per millilitre or to 0.1 mL of the prescribed
The precipitate obtained, after recrystaIlisation from hot solution add 0.1 mL of a 100 gIL solution of potassium
water R and drying in vacuo, has a melting point (2.2. 14) bromide R, 0.1 mL of a 20 gIL solution of resorcinol R
of 156 oC to 161 °C. and 3 mL of sulfun·c acid R. Heat on a water-bath for
5 min to 10 mino A dark-blue colour develops. AIlow to
Silicates cool and pour the solution into water R. The colour
Mix the prescribed quantity of the substance to be examined changes ro red.
in alead or platinum crucible by means of a copper wire
with about 10 mg of sodium fiuoride R and a few drops of Xanthines
sulfuric acid R to give a thin slurry. Cover the crucible with a To a few milligrams of the substance to be examined or the
thin, transparent plate of plastic under which a drop of prescribed quantity add 0.1 mL of strong hydrogen peroxide
water R is suspended and warm gently. Within a short time a solution R and 0.3 mL of dilute hydrochloric acid R . Heat ro
white ring is rapidly formed around the drop of water. dryness on a water-bath until a yeIlowish-red residue is
obtained. Add 0.1 mL of dilute ammonia R2. The colour of
Silver and Silver Compounds the residue changes to violet-red.
Dissolve about 10 mg of the substance to be examined in
10 mL of water R or use 10 mL of the prescribed solution. Zinc and Zinc Salts
Add 0.3 mL of hydrochloric acid R1. A curdled, white Dissolve 0.1 g o(the substance to be examined in 5 mL of
precipita te is formed that dissolves on addition of 3 mL of water R or use 5 mL of the prescribed solution. Add 0.2 mL
dilute ammonia R1. of strong sodium hydroxide solution R. A white precipitate is
formed. Add a further 2 mL of strong sodium hydroxide
Sodium and Sodium Salts
solution R . The precipitate dissolves. Add 10 mL of
A. Dissolve 0.1 g of the substance to be examined in 2 mL ammonium chloride solution R. The solution remains clear.
of water R or use 2 mL of the prescribed solution. Add 0.1 mL of sodium sulfide solution R. A flocculent white
Add 2 mL of a 150 gIL solution of potassium carbonate R precipitate is formed.
and heat to boiling. No precipitate is formed. Add 4 mL
of potassium pyroantimonate solution R and heat to boiling.
AIlow ro cool in iced water and if necessary rub the
2014 Appendix VII V-A259
solution (1 ppm NH,J R with 5 mL of water R and 0.3 mL of

Appendix VII alkaline potassium tetraiodomercurate solution R. Stopper the
test-tubes.
After 5 min, any yellow colour in the test solution is not
Limit Tests more intense than that in the standard.
METHODB
Nessler Cylinders In a 25 mL jar fitted with a cap, place the prescribed
Where the use of Nessler cylinders is prescribed in a test of the quantity of the finely powdered substance to be examined
Phannacopoeia, Nessler cylinders complying with the and dissolve or suspend in 1 mL of water R. Add 0.30 g of
following requirements should be used. heavy magnesium oxide R. Close irnmediately after placing a
Nessler cylinders comply with British Standard 612:1966 piece of sz1ver manganese paper R 5 mm square, wetted with a
(Specification for Nessler cylinders) . They are of c1ear glass few drops of water R, under the polyethylene cap . Swirl,
with a nominal capacity of 50 mL; the overall height is about avoiding projections of liquid, and allow to stand at 40 oC for
15 cm, the external height to the 50-mL mark 11.0 to 30 mino If the silver manganese paper shows a grey colour, it
12.4 cm, the thickness of the wall 1.0 to 1.5 mm and the is not more intense than that of a standard prepared at the
thickness of the base 1.0 to 3.0 mm. The external heights to same time and in the same manner using the prescribed
the 50-mL mark of cylinders used for a test must not differ volume of ammonium standard solution (1 ppm NH,J R, 1 mL
by more than 1 mm. of water R and 0.30 g of heavy magnesium oxide R .
Tubes for Comparative Tests Limit Test for Arsenic

(Ph. Eur. method 2.1.5) (Ph. Eur. method 2.4.2)
Tubes used for comparative tests are matched tubes of METHODA
colourless glass with a unifonn internal diameter. The base is The apparatus (see Figure 2.4.2.-1) consists of a 100 mL
transparent and flat.
conical flask c10sed with a ground-glass stopper through
A column of the liquid is examined down the vertical axis of which passes a glass tube about 200 mm long and of internal
the tube against a white background, or if necessary, against diameter 5 mm. The lower part of the tube is drawn to an
a black background. The examination is carried out in internal diameter of 1.0 mm, and 15 mm from its tip is a
diffused light. lateral orifice 2 mm to 3 mm in diameter. When the tube is
It is assumed that tubes with an internal diameter of 16 mm in position in the stopper, the lateral orifice should be at least
will be used. Tubes with a larger internal diameter may be 3 mm below the lower surface of the stopper. The upper end
used instead but the volume of liquid examined must then be of the tube has a perfectly fiat, ground surface at right angles
increased so that the depth of liquid in the tubes is not less to the axis of the tube. A second glass tube of the same
than where the prescribed volume of liquid and tubes 16 mm internal diameter and 30 mm long, with a similar fiat ground
in internal diameter are used. surface, is placed in contact with the first, and is held in
position by two spiral springs. Into the lower tube insert
Limit Test for Aluminium 50 mg to 60 mg of lead acetate caUon R, loosely packed, or a
(Ph. Eur. method 2.4. 17) small plug of cotton and a rolled piece of lead acetate paper R
Place the prescribed solution in a separating funnel and shake weighing 50 mg to 60 mg. Between the flat surfaces of the
with 2 quantities, each of 20 mL, and then with one 10 mL tubes place a disc or a small square of mereurie bromide
quantity of a 5 giL solution of hydroxyquinoline R in paper R large enough to cover the orifice of the tube (15 mm
chloroform R. Dilute the combined chloroform solutions to x 15 mm).
50.0 mL with chloroform R (test solution). In the conical fiask dissolve the prescribed quantity of the
Prepare a standard in the same manner using the prescribed substance to be examined in 25 mL of water R, or in the case
reference solution. of a solution adjust the prescribed volume to 25 mL with
Prepare a blank in the same manner using the prescribed water R . Add 15 mL of hydrochloric acid R, 0.1 mL of
blank solution. stannous ehloride solution R and 5 mL of potassium iodide
solution R , allow to stand for 15 min and introduce 5 g of
Measure the intensity ofthe fluorescence (2.2.21) ofthe test
activated zine R. Assemble the two parts of the apparatus
solution (11 ), of the standard (12) and of the blank (13) using
an excitant beam at 392 nm and a secondary filter with a immediately and irnmerse the fiask in a bath of water at a
transmission band 'centred on 518 nm or a monochromator temperature such that a unifonn evolution of gas is
set to transmit at this wavelength. maintained. Prepare a standard in the sarne manner, using
1 mL of arsenie standard solution (1 ppm As) R, diluted to
The fiuorescence (11 - 13 ) of the test solution is not greater 25 mL with water R.
than that of the standard (Ir h).
After not less than 2 h the stain produced on the mercuric
Limit Test for Ammonium bromide paper in the test is not more intense than that in the
(Ph. Eur. method 2.4.1) standard.
Unless otherwise prescribed, use method A. METHODB
METHODA Introduce the prescribed quantity of the substance to be
Dissolve the prescribed quantity of the substance to be examined into a test-tube containing 4 mL of hydrochlorie
examined in 14 mL of water R in a test-tube, make alkaline if acid R and about 5 mg of potassium iodide R and add 3 mL of
necessary by the addition of dilute sodium hydroxlde solution R hypophosphorous reagent R. Heat the mixture on a water-bath
and dilute to 15 mL with water R . To the solution add for 15 min, shaking occasionally. Prepare a standard in the
0.3 mL of alkaline potassium tetraiodomercurate solution R. same manner, using 0.5 mL of arsenic standard solution
(10 ppm As) R.
Prepare a standard by mixing 10 mL of ammonium standard
V-A260 Appendix VII 2014
After heating on the water-bath, any colour in the test Limit Test for Fluorides
solution is not more intense than that in the standard. (Ph. Eur. methad 2.4.5)
[----180 _ _
1....
o(")
~
Figure 2.4.2.-1. - Apparatus for limit test A for arsenic
30
o
o
..q-
Limit Test for Calcium

All solutions used for this test should be prepared with distilled
water R.
To 0.2 mL of alcoholic calcium standard solution
(100 ppm Ca) R, add 1 mL of ammonium oxalate solution R .
After 1 min, add a mixture of 1 mL of dilute acetic acid R and
15 mL of a solution comaining the prescribed quantity of the
substance ro be examined and shake. Prepare a standard in
the same manner using a mixture of 10 mL of aqueous
calcium standard solution (10 ppm Ca) R, 1 mL of dilute acetic
acid R and 5 mL of distilled water R. Figure 2.4.5.-1. - Apparatus for limit test for fluorides
After 15 min, any opalescence in the test solution is not
more intense than that in the standard. Dimensions in millimetres
Limit Test for ChIorides Introduce imo the inner tube of the apparatus (see Figure
2.4.5.-1) the prescribed quantity ofthe substance to be
(Ph. Eur. method 2.4.4) examined, 0.1 g of acid-washed sand R and 20 mL of a
To 15 mL of the prescribed solution add 1 mL of dilute nitn'c mixture of equal volumes of sulfuric acid R and water R. Heat
acid R and pour the mixture as a single addition into a test- the jacket containing tetrachlaraethane R maimained at its
tube comaining 1 mL of silver nitrate solution R2. Prepare a boiling poim (146 oC). Heat the steam generator and distil,
standard in the same manner using 10 mL of chlaride collecting the distillate in a 100 mL volumetric fiask
standard solution (5 ppm Cl) R and 5 mL of water R. Examine containing 0.3 mL of 0.1 M sadium hydraxide and 0.1 mL of
the tubes)aterally against a black background. phenolphthalein salutian R. Maintain a constant volume
After standing for 5 min protected from light, any (20 mL) in the tube during distillation and ensure that the
opalescence in the test solution is not more intense than that distillate remains alkaline, adding 0.1 M sadium hydroxide if
in the standard. necessary. Dilute the distillate to 100 mL with water R (test
solution). Prepare a standard in the same manner by
distillation, using 5 mL of fiuaride standard salution (10 ppm
F) R instead of the substance to be examined. Into two glass-
stoppered cylinders introduce 20 mL of the test solution and
20 mL of the standard and 5 mL of
aminamethylalizarindiacetic acid reagent R .
After 20 min, any blue colour in the test solution (originally If the result is difficult to judge, filter the solutions through a
red) is not more intense than that in the standard. suitable membrane filter (nominal pore size 0.45 ¡.1m). Carry
out the filtration slowly and uniforrnly, applying moderate
Limit Test for Heavy Metals and constant pressure to the piston. Compare the spots on
(Ph. Eur. method 2.4.8) the filters obtained with the different solutions.
The methods described below require the use of thioacetamide METHODC
reagent R. As an alternative, sodium sulfide solution Rl Test solution Place the prescribed quantity (not more
(0.1 mL) is usually suitable. Since tests prescribed in than 2 g) of the substance to be examined in a silica crucible
monographs have been developed using thioacetamide with 4 mL of a 250 gIL solution of magnesium sulfate R in
reagent R, if sodium sulfide solution Rl is used instead, it is dilute sulfuric acid R. Mix using a fine glass rod. Heat
necessary to inc1ude also for methods A, B and H a monitor cautiously. If the mixture is liquid, evaporate gendy to
solution, prepared from the quantity of the substance to be dryness on a water-bath. Progressively heat to ignition and
examined prescribed for the test, to which has been added continue heating until an almost white or at most greyish
the volume of lead standard solution prescribed for residue is obtained. Carry out the ignition at a temperature
preparation of the reference solution. The test is invalid if the not exceeding 800 oC . Allow to cool. Moisten the residue
monitor solution is not at least as intense as the reference with a few drops of dilute sulfuric acid R . Evaporate, ignite
solution. again and allow to cool. The total period of ignition must
METHODA not exceed 2 h. Take up the resídue in 2 quantities, each of
Test solution 12 mL of the prescribed aqueous solution of 5 mL, of dilute hydrochloric acid R. Add 0.1 mL of
the substance to be examined. phenolphthalein solution R, then concentrated ammonia R until a
pink colour is obtained. Cool, add glacial acetic acid R until
Reference solution (standard) A mixture of 10 mL of
the solution is decolorised and add 0.5 mL in excess. Filter
lead standard solution (1 ppm Pb) R or lead standard solution
(2 ppm Pb) R, as prescribed, and 2 mL of the prescribed
if necessary and wash the filter. Dilute to 20 mL with
water R.
aqueous solution of the substance to be examined.
Reference solution (standard) Prepare as described for
Blank solution A mixture of 10 mL of water R and 2 mL
the test solution, using the prescribed volume of lead standard
of the prescribed aqueous solution of the substance to be
solution (10 ppm Pb) R instead of the substance to be
examined.
examined. To 10 mL of the solution obtained add 2 mL of
To each solution, add 2 mL of buffer solution pH 3.5 R. the test solution.
Mix and add to 1.2 mL of thioacetamide reagent R.
Monitor solution Prepare as described for the test
Mix immediately. Examine the solutions after 2 mino
solution, adding to the substance to be examined the volume
System suitability The reference solution shows a slight of lead standard solution (10 ppm Pb) R prescribed for
brown colour compared to the blank solution. preparation of the reference solution. To 10 mL of the
Result Any brown colour in the test solution is not more solution obtained add 2 mL of the test solution.
intense than that in the reference solution. Blank solution A mixture of 10 mL of water R and 2 mL
If the result is difficult to judge, filter the solutions through a of the test solution.
suitable membrane filter (nominal pore size 0.45 ¡.1m). Carry To 12 mL of each solution, add 2 mL or'buffer solution
out the filtration slowly and uniforrnly, applying moderate pH 3.5 R. Mix and add to 1.2 mL of thioacetamide reagent R .
and constant pressure to the pis ton. Compare the spots on Mix immediately. Examine the solutions after 2 mino
the filters obtained with the different solutions.
System suitability
METHODB - the reference solution shows a slight brown colour
Test solution 12 mL of the prescribed solution of the compared to the blank solution,
substance to be examined prepared using an organic solvent - the monitor solution is at least as intense as the reference
containing a minimum percentage of water (for example, . solution.
dioxan containing 15 per cent of water or acetone containing
Result Any brown colour in the test solution is not more
15 per cent ofwater).
intense than that in the reference solution.
Reference solution (standard) A mixture of 10 mL of
lead standard solution (1 or 2 ppm Pb), as prescribed, and If the result is difficult to judge, filter the solutions through a
2 mL of the prescribed solution of the substance to be suitable membrane filter (nominal pore size 0.45 ¡.1m). Carry
examined in an organic solvent. Prepare the lead standard out the filtration slowly and uniforrnly, applying moderate
solution (1 or 2 ppm Pb) by dilution of lead standard solution and constant pressure to the piston. Compare the spots on
(100 ppm Pb) R with the solvent used for the substance to
be examined. METHODD
Blank solution A mixture of 10 mL of the solvent used Test solution In a silica crucible, mix thoroughly the
for the substance to be examined and 2 mL of the prescribed quantity of the substance to be examined with
prescribed solution of the substance to be exarnined in an 0.5 g of magnesium oxide Rl. Ignite to dull redness until a
organic solvent. homogeneous white or greyish-white mass is obtained.
To ~ach solution, add 2 mL of buffer solution pH 3.5 R. If after 30 min of ignition the mixture remains coloured,
Mix and add to 1.2 mL of thioacetamide reagent R. allow to cool, mix using a fine glass rod and repeat the
Mix immediate1y. Examine the solutions after 2 mino ignition. If necessary repeat the operation. Heat at 800 oC
for about 1 h. Take up the residue in 2 quantities, each of
System suitability The reference solution shows a slight
5 mL, of a mixture of equal volumes of hydrochloric acid Rl
brown colour compared to the blank solution.
and water R. Add 0.1 mL of phenolphthalein solution R and
Result Any brown colour in the test solution is not more then concentrated ammonia R until a pink colour is obtained.
intense than that in the reference solution.
Membrane
Prefilter filter
1.... : ... ·.... ;·.·.: ... ·.... ;·.·.: .. 1
fr··· / .·· .; . . ;. . . '(."1'
Membrane Prefilter
filter
ex) Prefiltration Filtration of the

ex) of the solution solution after
T""" Joint
(Method E) addition of the
reagents
(Method E)
13
15.7
17
Figure 2.4.8.-1. - Apparatus for the test for heavy metals
Cool, add glacial acetic acid R until the solution is decolorised Reference solution (standard) Unless otherwise
and add 0.5 mL in excess. Filter if necessary and wash the prescribed, dilute the prescribed volume of lead standard
filter. Dilute 10 20 mL with water R. solution (J ppm Pb) R to the same volume as the test
Reference solution (standard) Prepare as described for solution.
the test solution using the prescribed volume of lead standard Prepare the filtration apparatus by adapting the barrel of a
solution (J O ppm Pb) R instead of the substance to be 50 mL syringe without its piston to a support containing, on
examined and drying in an oven at 100-105 oc. To 10 mL the plate, a membrane filter (nominal pore size 3 ~lm) and
of the solution obtained add 2 mL of the test solution. 'aboye it a prefilter (Figure 2.4.8.-1).
Monitor solution Prepare as described for the test Transfer the test solution into the syringe barrel, put the
solution, adding 10 the substance to be exarnined the volume piston in place and then apply an even pressure on it until
of lead standard solution (JO ppm Pb) R prescribed for the whole of the liquid has been filtered. In opening the
preparation of the reference solution and drying in an oven support and rerooving the prefilter, check that the membrane
at 100-105 oC. To 10 mL of the solution obtained add filter remains uncontaroinated with impurities. If this is not
2 mL of the test solution. the case replace it with another membrane filter and repeat
Blank solution A mixture of 10 roL of water R and 2 mL the operation under the same conditions.
of the test solution. To the prefiltrate or to the prescribed volume of the
To 12 mL of each solution, add 2 mL of buffer solution prefiltrate add 2 mL of buffer solution pH 3.5 R . Mix and add
pH 3.5 R . Mix and add 10 1.2 mL of thioacetamide reagent R . to 1.2 mL of thioacetamide reagent R. Mix irnmediately and
Mix irnmediately. Examine the solutions after 2 mino allow to stand for 10 min and again filter as described aboye,
System suitability but inverting the order of the filters, the liquid passing first
through the membrane filter before passing through the
- the reference solution shows a slight brown colour prefilter (Figure 2.4.8 .-1). The filtration must be carried out
compared to the blank solution, slowly and uniforrnly by applying moderate and constant
- the monitor solution is at least as intense as the reference pressure 10 the pis10n of the syringe. After complete
solution. filtration, open the support, remove the membrane filter, and
Result Any brown colour in the test solution is not more dry using filter papero
intense than that in the reference solution. In parallel, treat the reference solution in the same manner as
If the result is difficult 10 judge, filter the solutions through a the test solution.
suitable membrane filter (nominal pore size 0.45 ¡lm). Carry Result The colour of the spot obtained with the test
out the filtration slowly and uniforrnly, applying moderate solution is not more in tense than that obtained with the
and constant pressure to the pis ton. Compare the spots on reference solution.
METHODF
METHODE Test solution Place the prescribed quantity or volume of
Test solution Dissolve the prescribed quantity of the the substance 10 be examined in a c1ean, dry, 100 mL long-
substance 10 be examined in 30 mL of water R or the necked combustion flask (a 300 roL flask may be used if the
prescribed volume. reaction foaros excessively). Clamp the flask at an angle of
45°. If the substance to be exaroined is a solid, add a
sufficient volume of a mixture of 8 mL of sulfuric acid R and Test solution Place the prescribed amount of the
10 mL of nitric acid R to moisten the substance thoroughly; substance to be examined (not more than 0.5 g) in a
if the substance to be examined is a liquid, add a few suitable, c1ean beaker. Add successively 2.7 mL of sulfuric
millilitres of a mixture of 8 mL of sulfuric acid R and 10 mL acid R, 3.3 mL of niuic acid R and 2.0 mL of strong hydrogen
of nitric acid R. Warrn gently until the reaction commences, peroxide solution R using a magnetic stirrer. AlIow the
allow the reaction to subside and add additional portions of substance to react with a reagent before adding the next one.
the same acid mixture, heating after each addition, until a Transfer the mixture to a dry high-pressure-resistant
total of 18 mL of the acid mixture has been added. Increase digestion vessel (fiuoropolymer or quartz glass).
the amount of heat and boil gently until the solution Reference solution (standard) Prepare as described for
darkens. Cool, add 2 mL of nitric acid R and heat again until the test solution, using the prescribed volume of lead standard
the solution darkens. Continue the heating, followed by the solution (lO ppm Pb) R instead of the substance to be
addition of nitric acid R until no further darkening occurs, examined.
then heat strongly until dense, white fumes are produced. Monitor solution Prepare as prescribed for the test
Cool, cautiously add 5 mL of water R, boil gently until solution, adding to the substance to be examined the volume
dense, white fumes are produced and continue heating to of lead standard solution (la ppm Pb) R prescribed for the
reduce to 2-3 mL. Cool, cautiously add 5 mL of water R preparation of the reference solution.
and examine the colour of the solution. If the colour is
Blank solution Prepare as described for the test solution,
yellow, cautiously add 1 mL of strong hydrogen peroxide
solution R and again evaporate until dense, white fumes are omitting the substance to be examined.
produced and reduce to a volume of 2-3 mL. If the solution Close the vessels and place in a laboratory microwave oven.
is still yellow in colour, repeat the addition of 5 mL of Digest using a sequence of 2 separate suitable programmes.
water R and 1 mL of strong hydrogen peroxide solution R until Design the programmes in several steps in order to control
the solution is colourless. Cool, dilute cautiously with the reaction, monitoring pressure, temperature or energy
water R and rinse into a 50 mL colour comparison tube, depending on the type of microwave oven available. After the
ensuring that the total volume do es not exceed 25 mL. first programme allow the digestion vessels to cool before
Adjust the solution to pH 3.0-4.0, using short range pH opening. Add to each vessel 2.0 mL of strong hydrogen
indicator paper as external indicator, with concentrated peroxlde solution R and digest using the second programme.
ammonia Rl (dilute ammonia Rl may be used, if desired, as After the second programme allow the digestion vessels to
the specified range is approached), dilute with water R to cool before opening. If necessary to obtain a c1ear solution,
40 mL and mix. Add 2 mL of buffer solution pH 3.5 R. repeat the addition of strong hydrogen peroxide solution R and
Mix and add to 1.2 mL of thioacetamide reagent R. the second digestion programme.
Mix immediately. Dilute to 50 mL with water R and mix. Cool, dilute cautiously with waterR and rinse into a fiask,
Reference soludon (standard) Prepare at the same time ensuring that the total volume do es nor exceed 25 mL.
and in the same manner as the test solution, using the Using short-range pH indicator paper as external indicator,
prescribed volume of lead standard solution (lO ppm Pb) R. adjust the solutions to pH 3.0-4.0 with concentrated
Monitor solution Prepare as described for the test ammonia Rl (dilute ammonia Rl may be used as the specified
solution, adding to the substance to be examined the vo)ume range is approached). To avoid heating of the solutions use
of lead standard solution (la ppm Pb) R prescribed for the an ice-bath and a magnetic stirrer. Dilute to 40 mL with
preparation of the reference solution. water R and mix. Add 2 mL of buffer solution pH 3.5 R.
Blank solution Prepare as described for the test solution, Mix and add to 1.2 mL of thioacetamide reagent R.
omitting the substance to be examined. Mix immediately. Dilute to 50 mL with water R, mix and
allow to stand for 2 mino
Examine the solutions vertically against a white background
Filter the solutions through a suitable membrane filter
after 2 mino
(nominal pore size 0.45 ¡.1m). Carry out the filtration slowly
System suitability and uniforrnly, applying moderate and constant pressure to
- the reference solution shows a brown colour compared to the piston. Compare the spots on the filters obtained with
the blank solution, the different solutions.
- the monitor solution is at least as intense as the reference System suitability
solution. - the spot obtained with the reference solution shows a
Result Any brown colour in the test solution is not more brown colour compared to the spot obtained with the
intense than that in the reference solution. blank solution,
If the result is difficult to judge, filter the solutions through a - the spot obtained with the monitor solution is at least as
suitable membrane filter (nominal pore size 0.45 ¡.1m). Carry intense as the spot obtained with the reference solution.
out the filtration slowly and uniforrnly, applying moderate Result The brown colour of the spot obtained with the
and constant pressure to the piston. Compare the spots on test solution is not more intense than that of the spot
the filters obtained with the different solutions. obtained with the reference solution.
METHODG METHODH
CAUTION: when using high-pressure digestion vessels the safety Test solution Dissolve the prescribed quantity of the
precautions and operating instructions given by the manufacturer substance to be examined in 20 mL of the solvent or solvent
must be followed. The digestion cycles have to be elaborated mixtureprescribed.
depending on the type of murowave oven to be used (for example,
Reference solution Dilute the prescribed volume of lead
energy-controlled microwave ovens, temperature-controlled
standard solution (lO ppm Pb) R to 20 mL with the solvent or
microwave ovens or high-pressure ovens). The cycle must conform
to the manufacturer's instructions. The digestion cycle is suitable If
solvent mixture prescribed.
a clear solution is obtained.
Blank solution 20 mL of the solvent or solvent mixture the solution of hydroxyquinoline in chloroform, shake for
prescribed. 1 min, allow to stand and separate. Use the lower layer for
To each solution, add 2 mL of buffer solution pH 3.5 R . Mix. comparison. Prepare a standard in the same manner using a
(In some cases precipitation occurs, in which case the specific mixture of 1 mL of magnesium standard solution
monograph would describe re-dissolution in a defined volume of a (10 ppm Mg) R and 9 mL of water R.
given solvent.) Add to 1.2 mL of thioacetamide reagent R. Any colour in the solution obtained from the substance to be
Mix immediately and allow to stand for 2 mino Filter the examined is not more intense than that in the standard.
solutions through a suitable membrane filter (nominal pore
size 0.45 Jlm). Compare the spots on the filters obtained with Limit Test for Magnesium and AIkaline-earth
the different solutions. Metals
System suitability The spot obtained with the reference (Ph. Eur. method 2.4.7)
solution shows a brownish-black colour compared to the spot To 200 mL of water R add 0.1 g of hydroxylamine
obtained with the blank solution. hydrochloride R, 10 mL of ammonium chloride buffer solution
Result The brownish-black colour of the spot obtained pH 10. O R, 1 mL of 0.1 M zinc sulfate and about 15 mg of
with the test solution is not more intense than that of the mordant black 11 tritura te R. Heat to about 40 oC. Titrate
spot obtained with the reference solution. with 0.01 M sodiUln edetate until the violet colour changes to
full blue. To the solution add the prescribed quantity of the
Limit Test for Iron substance to be examined dissolved in 100 mL of water R or
(Ph. Eur. method 2.4.9) use the prescribed solution. If the colour of the solution
Dissolve the prescribed quantity of the substance to be changes to violet, titrate with 0.01 M sodium edetate until the
examined in water R and dilute to 10 mL with the same full blue colour is again obtained .
solvent or use 10 mL of the prescribed solution. Add 2 mL The volume of o. 01 M sodium edetate used in the second
of a 200 giL solution of citric acid R and 0.1 mL of titration does not exceed the prescribed quantity.
thioglycollic acid R. Mix, make alkaline with ammonia R and
dilute to 20 mL with water R. Prepare a standard in the same Limit Test for Heavy Metals in Herbal Drugs and
manner, using 10 mL of iron standard solution (1 ppm Fe) R. Fatty Oils
After 5 min, any pink colour in the test solution is not more (Ph. Eur. method 2.4.2 7)
intense than that in the standard . Examine by atomic absorption spectrometry (2.2.23).
CAUTION: when using closed high-pressure digestion vessels and
Limit Test for Lead in Sugars microwave laboratory equipment, be famz1iar with the safety and
(Ph. Eur. method 2.4.10) operating instructions given by the manufacturero
Determine the lead by atomic absorption spectrometry Apparatus
(2.2.23, Method lI) .
The apparatus typically consists of the following:
Test solution Dissolve 20.0 g of the substance to be
- as digestion flasks, polytetrafluoroethylene flasks with a
examined in a mixture of equal volumes of dilute acetic acid R
volume of about 120 mL, fitted with an airtight cJosure, a
and water R and dilute to 100.0 mL with the same mixture
valve to adjust the pressure inside the container and a
of solvents. Add 2.0 mL of a cJear 10 gIL solution of
polytetrafluoroethylene tube to allow release of gas,
ammonium pyrrolidinedithiocarbamate R and 10.O mL of methyl
isobutyl ketone R and then shake for 30 s protected from - a system to make flasks airtight, using the same torsional
bright light. Allow the layers to separate and use the methyl force for each of them,
isobutyl ketone layer. - a microwave oven, with a magnetron frequency of
Reference solutions. Prepare 3 reference solutions in the 2450 MHz, with a selectable output from O to 630 ± 70
same manner as the test solution but adding 0.5 mL, W in 1 per cent increments, a programmable digital
1.0 mL and 1.5 mL respectively of lead standard solution computer, a polytetrafluoroethylene-coated microwave
(10 ppm Pb) R in addition to the 20.0 g of the substance to cavity with a variable speed exhaust fan, a rotating
be examined. turntable drive system and exhaust tubing to vent fumes,
Set the zero of the instrument using methyl isobutyl ketone R - an atomic absorption spectrometer, equipped with hollow-
treated as described for the test solution without the cathode lamps as source of radiation and a deuterium
substance to be examined. Measure the absorbance at lamp as background corrector; the system is fitted with:
283.3 nm using alead hollow-cathode lamp as source of (a) a graphite furnace as atomisation device for cadmium,
radiation and an air-acetylene flameo copper, iron, lead, nickel and zinc.
The substance to be examined contains not more than (b) an automated continuous-flow hydride vapour
0.5 ppm of lead, unless otherwise prescribed. generation system for arsenic and mercury.
Limit Test for Magnesium Method

In case alternative apparatus is used, an adjustment of the
instrument parameters may be necessary.
To 10 mL of the prescribed solution add 0.1 g of disodium
tetraborate R. Adjust the solution, if necessary, to pH 8.8 to Clean all the glassware and laboratory equipment with a
pH 9.2 using dz1ute hydrochloric acid R or dilute sodium 10 gIL solution of nitric acid R before use.
hydroxide solution R . Shake with 2 quantities, each of 5 mL, Test solution In a digestion flask place the prescribed
of a 1 giL solution of hydroxyquinoline R in chloroform R, for quantity of the substance to be examined (about 0.50 g of
1 min each time. Allow to stand. Separate and discard the powdered drug (1400) (2.9.12) or 0.50 g offatty oil).
organic layer. To the aqueous solution add 0.4 mL of Add 6 mL of heavy metal-free nitric acid R and 4 mL of
butylamine R and 0.1 mL of triethanolamine R. Adjust the heavy metal-free hydrochloric acid R. Make the flask airtight.
solution, if necessary, to pH 10.5 to pH 11.5. Add 4 mL 9f
Place the digestion fiasks in the microwave oven. Carry out Acid reagent A 515 gIL solution of heavy metal-free
the digestion in 3 steps according to the following hydrochloric acid R.
programme, used for 7 fiasks each containing the test Reducing reagent A 10 gIL solution of stannous chloride R
solution: 80 per cent power for 15 min, 100 per cent power in dilute heavy metal-free hydrochloric acid R.
for 5 min, 80 per cent power for 20 mino The instrumental parameters in Table 2.4 .27.-2 may be
At the end of the cyc1e allow the fiasks to cool in air and to used.
each add 4 mL of heavy metal-free sulfun'c acid R. Repeat the
digestion programme. After cooling in air, open each Table 2.4.27.-2
digestion fiask and introduce the c1ear, colourless solution
As Hg
obtained into a 50 mL volumetric fiask. Rinse each digestion
fiask with 2 quantities, each of 15 mL, of water R and collect Wavelength nm 193.7 253.7
the rinsings in the volumetric fiask. Add l.0 mL of a 10 gIL Slit width nm 0.2 0.5
solution of magnesium nitrate R and 1.0 mL of a 100 gIL
Lamp current mA 10 4
solution of am1110nium dihydrogen phosphate R and dilute to
50.0 mL with water R. Acid reagent f10w rate mL/ min 1.0 1.0
Blank solution Mix 6 mL of heavy metal-free nitric acid R Reducing reagent flow rate mL/ min 1.0 1.0
and 4 mL of heavy metal-free hydrochlon'c acid R in a Sample solution f10w rate mL/min 7.0 7.0
digestion fiask. Carry out the digestion in the same manner
as for the test solution. Absorption cell Quartz Quartz
(heated) (unheated)
CADMIUM, COPPER, IRON, LEAD, NICKEL AND ZINC Background corrector off off
Measure the content of cadmium, copper, iron, lead, nickel Nitrogen f10w rate L/ min 0.1 0.1
and zinc by the standard additions method (2.2.23,
Method Il), using reference solutions of each heavy metal and
the instrumental parameters described in Table 2.4.27.-l.
Limit Test for Nicke1 in PolyoIs
The absorbance value of the blank solution is automatically
subtracted from the value obtained with the test solution.
Determine the nickel by atomic absorption spectrometry
(2.2.23, M ethod Il).
Table 2.4.27.-1
Test solution Dissolve 20.0 g of the substance to be
Cd Cu Fe Ni Pb Zn
examined in a mixture of equal volumes of di/ute acetic acid R
Wavelength nm 228.8 324.8 248.3 232 283.5 213.9 and water R and dilute to 100.0 mL with the same mixture
Slit width nm 0.5 0.5 0.2 0.2 0.5 0.5 of solvents. Add 2.0 mL of a saturated solution of ammonium
pyn'Olidinedithiocarbamate R (about 10 gIL) and 10.0 mL of
Lamp current mA 6 7 5 10 5 7
methyl isobut:yl kecone R and then shake for 30 s protected
Ignition oC 800 800 800 800 800 800 from bright light. Allow the layers to separate and use the
temperature
Atomisation oC 1800 2300 2300 2500 2200 2000
methyl isobutyl ketone layer.
temperature Referen.ce solutions. Prepare 3 reference solutions in the
Background on off off off off off
corrector
same manner as the test solution but adding 0.5 mL,
Nitrogen flow L/ min 3 3 3 3 3 LO mL and l.5 mL respectively of nickel standard solution
(10 ppm Ni) R in addition to the 20.0 g ofthe substance to
be examined.
ARSENIC AND MERCURY Set the zero of the instrument using methyl isobut:yl kecone R
Measure the content of arsenic and mercury in comparison treated as described for preparation of the test solution
with the reference solutions of arsenic or mercury at a known omiuing the substance to be examined. Measure the
concentration by direct calibration (2.2.23, M ethod l) using absorbance at 232.0 nm using a nickel hollow-cathode lamp
an automated continuous-fiow hydride vapour generation as source of radiation and an air-acetylene fiame .
system. Tne substance to be examined contains not more than
The absorbance value of the blank solution is automatically 1 ppm of nickel, unless otherwise prescribed.
subtracted from the value obtained with the test solution.
Limit Test for Phosphates
ARSENIC
Sample solution To 19.0 mL of the test solution or of (Ph. Eur. method 2.4.11)
the blank solution as prescribed aboye, add 1 mL of a To 100 mL ofthe solution prepared and, ifnecessary,
200 gIL solution of potassiwn iodide R. AlIow the test solution neutralised as prescribed add 4 mL of sulfomolybdic
to stand at room temperature for about 50 min or at 70 oC reagent R3. Shake and add 0.1 mL of stanllOUS chloride
for about 4 mino solution R1. Prepare a standard in the same manner using
2 mL of phosphate standard solution (5 ppm PO,J R and
Acid reagent H eavy metal-free hydrochloric acid R.
98 mL of water R. After 10 min, compare the colours using
Reducing reagent A 6 giL solution of sodium 20 mL of each solution.
tetrahydroborate R in a 5 gIL solution of sodium hydroxlde R .
Any colour in the test solution is not more intense than that
The instrumental parameters in Table 2.4.27.-2 may be in the standard.
used.
MERCURY Limit Test for Potassium
Sample solution Test solution or blank solution, as (Ph . Eur. method 2.4.12)
prescribed aboye. To 10 mL of the prescribed solution add 2 mL of a freshly
prepared 10 gIL solution of sodium tetraphenylborate R.
Prepare a standard in the same manner using a mixture of

5 mL of potassium standard solution (20 ppm K) R and 5 mL
of water R.
After 5 min, any opalescence in the test solution is not more
intense than that in the standard.
Limit Test for Sulfates
All solutions used for this test must be prepared with distilled
water R.
Add 3 mL of a 250 gIL solution of barium eh/oride R to
4.5 mL of sulfate standard solution (lO ppm SO,J R1. Shake
and allow to stand for 1 mino To 2.5 mL of this suspension,
add 15 mL of the solution to be examined and 0.5 mL of
aeetie acid R . Prepare a standard in the same manner using
15 mL of sulfate standard solution (lO ppm SO,J R instead of
the solution to be examined.
After 5 min, any opalescence in the test solution is not more
intense than that in the standard.
2014 Appendix VIII B V-A267
disc electro de or a carbon electrode) and a reference

Appendix VIII electrode (for example, a calomel electro de or a silver-silver
chloride electrode).
A three-electrode apparatus is sometimes used, consisting of
A. Non-aqueous Titration an indicator electro de, a reference electro de and a polarised
(No Ph. Eur. methad) auxiliary electrode.
METHOD 1 Method Set the potential of the indicator electrode as
Dissolve the prescribed quantity of the substance being prescribed and plot a graph of the initial current and the
examined in a suitable volume of anhydrous acetic acid values obtained during the titration as functions of the
previously neutralised using the indicator specified in the quantity of titrant added. Add the titrant in not fewer than 3
monograph, warming and cooling if necessary, or prepare a successive quantities equal to a total of about 80 per cent of
solution as directed. When the substance is a salt of the theoretical volume corresponding to the presumed
hydrochloric or hydrobromic acid, add 15 mL of mercury(1I) equivalence point. The 3 values must fall on a straight lineo
acetate solution before neutralising the solvent, unless Continue adding the titrant beyond the presumed
otherwise directed in the monograph. Titrate with equivalence point in not fewer than 3 successive quantities.
O.IM perchloric acid VS to the colour change of the indicator The values obtained must fall on a straight lineo The point of
that corresponds to the maximum absolute value of dE/dV intersection of the 2 lines represents the end-point of the
(where E is the electromotive force and Vis the volume of titration.
titrant) in a potentiometric titration, Appendix VIII B, of the For amperometric titration with 2 indicator electrodes, the
substance being examined. The indicator specified in the whole titration curve is recorded and used to determine the
monograph is also used for the neutralisation of the end-point.
mercury(lI) acetate solution and the standardisation of the
titrant. Potentiometric Titration
When the temperature (t2) of the titrant at the time of the (Ph. Eur. method 2.2.20)
assay differs from the temperature (ti) of the titrant when it In a potentiometric titration the end-point of the titration is
was standardised, multiply the volume of the titrant required determined by following the variation of the potential
by [1+0 .0011 (t1 - t2)] and calculate the result ofthe assay difference between 2 electrodes (either one indicator
from the corrected volume. electrode and one reference electrode or 2 indicator
Carry out a blank titration when necessary. electrodes) immersed in the solution to be examined as a
METHOD II function of the quantity of titrant added.
The titrant, solvent and, where necessary, the indicator to be The potential is usually measured at zero or practically zero
used are stated in the monograph. current.
Protect the solution and titrant from atrnospheric carbon Apparatus The apparatus used (a simple potentiometer or
dioxide and moisture throughout the determination. electronic device) comprises a voltrneter allowing readings to
the nearest millivolt.
Dissolve the substance being examined in a suitable volume
of the solvent previously neutralised to the indicator, The indicator electrode to be used depends on the substance
warming and cooling if necessary, or prepare a solution as to be determined and may be a glass or metal electrode (for
directed. Titrate to the colour change of the indicator that example, platinum, gold, silver or mercury). The reference
corresponds to the maximum absolute value of dE/dV (where electrode is generally a calomel or a silver-silver chloride
E is the electromotive force and Vis the volume of titrant) in electrode .
a potentiometric titration, Appendix VIII B, of the substance For acid-base titrations and unless otherwise prescribed, a
under examination. The titrant is standardised using the glass-calomel or glass-silver-silver chloride electrode
same solvent and indicator as specified for the substance. combination is used.
Carry out a blank titration when necessary. Method In potentiometric titrations of weak acids or bases
using non-aqueous solvents, either carry out a blank
determination or pre-neutralise the solvent mixture, if
necessary, before dissolution of the substance to be
B. Amperometric, Potentiometric and examined. Where it is impracticable to use potentiometric
detection for this purpose, the solvent mixture can be pre-
Voltametric Titrations neutralised by titration using a suitable indicator. Sorne
Amperometric Titration examples are given below:

In amperometric titration the end-point is determined by Titrant Indicator
following the variation of the current measured between Perchloric acid Cryslal violel solulion R
2 electrodes (either one indicator electrode and one reference
Tetrabutylammonium hydroxide 3 gjL solution of Ihymol blue R
electrode or 2 indicator electrodes) irnmersed in the solution in melhanol R
to be examined and maintained at a constant potential Ethanolic sodium hydroxide Thymolphlhalein solulion R
difference as a function of the quantity of titrant added.
The potential of the measuring electrode is sufficient to
Plot a graph of the variation of potential difference as a
ensure a diffusion current for the electroactive substance.
function of the quantity of the titrant added, continuing the
Apparatus The apparatus comprises an adjustable voltage addition of the titrant beyond the presumed equivalence
source and a sensitive microammeter; the detection system point. The end-point corresponds to a sharp variation of
generally consists of an indicator electrode (for example, a potential difference.
platinum electro de, a dropping-mercury electro de, a rotating-
V-A268 Appendix VIII e 2014
Voltametric Titrations Cool and after about 5 min, unless otherwise prescribed,
(Ph. Eur. method 2.2.65) carefully unstopper the fiask. W ash the ground parts and the
In voltametric titration the end-point of the titration is walls of the fiask, as well as the sample carríer, with water R.
determined by following the variation of the voltage Combine the combustion products and the washings and
measured between 2 electrodes (either 1 indicator electrode proceed as prescribed in the monograph.
and 1 reference electrode or 2 indicator electrodes) immersed
For Iodine
in the solution to be examÍned and maintained at a constant
current as a function of the quantity of titrant added. (No Ph. Eur. method)
Bum the specified quantity of the substance being examined
Apparatus The apparatus comprises an adjustable current
in the prescribed manner using a mixture of 10 mL of water
source and a voltmeter; the detection system generally
and 2 mL of 1M sodium hydroxide as the absorbing liquido
consists of an indicator electro de (for example, a platinum
When the process is complete, add to the fiask an excess of
electro de, a rotating-disc electrode or a carbon electro de)
acetic bromine solution (between 5 and 10 mL) and allow to
and a 2n d electrode (for example, a platinum electro de, a
stand for 2 minutes. Remove the excess of bromine by the
rotating-disc electrode or a carbon electrode).
addition ofjonnic acid (about 0.5 10 I mL), rinse the sides of
Method Set the current to the indicator electrode as the fiask with water and displace any bromine vapour aboye
prescribed in the monograph and plot a graph of the initial the liquid with a current of air. Add 1 g of potassium iodide
voltage and the values obtained during the titration as and titrate with 0.02M sodium thiosulfate VS using starch
functions of the quantity of titrant added. Add the titrant in mucilage, added towards the end of the titration, as indicator.
not fewer than 3 successive quantities equal to a total of Each mL of 0 .02M sodium thiosulfate VS is equivalent to
about 80 per cent of the theoretical volume corresponding to 0.4230 mg 0fI.
the presumed equivalence point. The 3 values must fall on a
straight lineo Continue adding the titrant beyond the
presumed equivalence point in not fewer than 3 successive
quantities. The values obtained must fall on another straight D. Complexometric Titrations
lineo The point of intersection of the 2 lines represents the
end-point of the titration.
Using titration systems for voltametric titration with 2 Aluminium
indicator electro des, the whole titration curve is recorded and Introduce 20.0 mL of the prescribed solution into a 500 mL
used to determine the end-point. conical fiask, add 25 .0 mL of 0. 1 M sodium edetate and
10 mL of a mixture of equal volumes of a 155 giL solution
Detenrunation of Primary Aromatic Amino-
of ammonium acetate R and dilute acetic acid R. Boil for 2 min,
nitro gen
then cool. Add 50 mL of ethanol R and 3 mL of a freshly
(Ph. Eur. method 2.5.8) prepared 0.25 gIL solution of dithizone R in ethanol R. Titrate
Dissolve the prescribed quantity of the substance 10 be the excess of sodium edetate with 0.1 M zinc sulfate until the
examined in 50 mL of dilute hydrochloric acid R or in another colour changes from greenish-blue to reddish-violet.
prescribed solvent and add 3 g of potassium bromide R. Cool 1 mL of 0. 1 M sodium edetate is equivalent to 2.698 m g of
in ice-water and titrate by slowly adding 0.1 M sodium nitrite Al.
with constant stirring.
Determine the end-point electrometrically or by the use of Bismuth
the prescribed indicator. Introduce the prescribed solution into a 500 mL conical
fiask. Dilute 10 250 mL with water R and then, unless
otherwise prescribed, add dropwise, with shaking, concentrated
ammonia R until the mixture becomes cloudy. Add 0.5 mL
C. Oxygen-flask Combustion of nitric acid R. Heat to about 70 oC until the cloudiness
(Ph. Eur. method 2.5.10) disappears completely. Add about 50 mg of xylenol orange
Unless otherwise prescribed the combustion fiask is a conical triturate R and titrate with 0.1 M sodium edetate until the
fiask of at least 500 mL capacity of borosilicate glass with a colour changes from pinkish-violet to yellow.
ground-glass stopper fitted with a suitable carrier for the 1 mL of 0. 1 M sodiu m edetate is equivalent to 20.90 mg of Bi.
sample, for example in platinum or platinum-iridium .
Calcium
Finely grind the substance to be examined, place the
prescribed quantity in the centre of a piece of filter paper Introduce the prescribed solution into a 500 mL conical
measuring about 30 mm by 40 mm provided with a small fiask, and dilute to 300 mL with water R. Add 6.0 mL of
strip about 10 mm wide and 30 mm long. If paper strong sodium hydroxide solution R and about 15 mg of
impregnated with lithium carbonate is prescribed, moisten calconecarboxylic acid triturate R . Titrate with 0.1 M sodium
the centre of the paper with a saturated solution of lithium edetate until the colour changes from violet to full blue.
carbonate R and dry in an oven before use. Envelop the 1 mL of 0.1 M sodium edetate is equivalent 10 4.008 mg of
substance to be examined in the paper and place it in the Ca.
sample carríer. Introduce into the fiask water R or the
prescribed solution designed to absorb the combustion Magnesium
products, displace the air with oxygen by means of a tube Introduce the prescribed solution into a 500 mL conical fiask
having its end just aboye the liquid, moisten the neck of the and dilute to 300 mL with water R . Add 10 mL of
fiask with water R and close with its stopper. Ignite the paper ammonium chloride buffer solution pH 10. O R and about 50 mg
strip by suitable means with the usual precautions . Keep the of mordant black 11 triturate R. Heat to about 40 oC then
fiask firmly closed during the combustion. Shake the fiask titrate at this temperature with 0. 1 M sodium edetate until the
vigorously to completely dissolve the combustion products. colour changes fro m violet to full blue.
2014 Appendix VIII E V-A269
1 mL of 0.1 M sodium edetate is equivalent ro 2.431 mg of Ion-selective electro des may be primary electrodes with a
Mg. crystal or non-crystal membrane or with a rigid matrix (for
example, glass electrodes), or electro des with charged
Lead (positive or negative) or uncharged mobile carriers, or
Introduce the prescribed solution into a 500 mL conical flask sensitised electrodes (enzymatic-substrate electro des, gas-
and dilute to 200 mL with water R. Add about 50 mg of indicaror electrodes). The reference electro de is generally a
xylenol orange tritura te R and hexamethylenetetramine R until silver-silver chloride electrode or a calomel electro de, with
the solution becomes violet-pink. Titrate with 0.1 M sodium suitable junction liquids producing no interference.
edetate until the violet-pink colour changes to yellow. Procedure Carry out each measurement at a temperature
1 mL of 0.1 M sodium edetate is equivalent to 20 .72 mg of constant to ± 0.5 oC, taking into account the variation of
Pb. the slope of the electrode with temperature (see
Table 2.2.36.-1). Adjust the ionic strength and possibly the
Zinc pH of the solution to be analysed using the buffer reagent
Introduce the prescribed solution into a 500 mL conical flask described in the monograph and equilibra te the electrode by
and dilute to 200 mL with water R. Add about 50 mg of immersing it in the solution ro be analysed, under slow and
xylenol orange tritura te R and hexamethylenetetramine R until unifortn stirring, until a constant reading is obtained.
the solution becomes violet-pink. Add 2 g of
hexamethylenetetramine R in excess . Titrate with 0.1 M sodium Table 2.2.36.-1. - Values of k at different temperatures
edetate until the violet-pink colour changes to yellow. Temperature (OC) k
1 mL of 0.1 M sodium edetate is equivalent ro 6.54 mg of Zn.
20 0.0582
25 0.0592
30 0.0602
E. Potentiometric Determination of lonic
Concentration Using lon-selective If the electro de system is used frequently, check regularly the
Electrodes repeatability and the stability of responses, and the linearity
of the calibration curve or the calculation algorithm in the
(Ph. Eur. method 2.2.36) range of concentrations of the test solution¡ if not, carry out
Ideally, the potential E of an ion-selective electrode varies the test before each set of measurements. The response of
linearly with the logarithm of the activity a¡ of a given ion, as the electro de may be regarded as linear if the slope S of the
expressed by the Nemst equation: calibration curve is approximately equal to klzi, per unit of
pC¡.
RT
E = Ea + 2.303 -F logai
Zi Method 1 (direct calibration)
Measure ar least three times in succession the potential of at
Ea pan of the constant potential due to the apparatus
least three reference solutions spanning the expected
used,
concentration of the test solution. Calculate the calibration
R gas constant,
curve, or plot on a chart the mean potential E obtained
T absolute temperature,
against rhe concentrarion of the ion to be derertnined
F Faraday's number,
expressed as - log C¡ or pC¡.
Z¡ charge number of the ion including its signo
Prepare the test solution as prescribed in the monographi
At a constant ionic strength, the following holds: measure the potential three times and, from the mean
potential, calculate the concentration of the ion to be
k
E = Ea + -logfGi
Z¡ ,
detertnined using the calibration curve.
Method 11 (multiple standard additions)

molar concentration of the ion, Prepare the test solution as prescribed in the monograph.
the activity coefficient (a¡ == JCD, Measure the potential at equilibrium ET of a volume VT of
RT this solution of unknown concentration CT of the ion ro be
k detertnined. Make at least three consecutive addirions of a
F
volume Vs negligible compared ro VT (Vs ~ 0.01 VT ) of a
If: Ea + ~ log] = Eb and S = k reference solution of a concentration Cs known ro be within
Z¡ Zi the linear pan of the calibration curve. After each addirion,
S = slope of the calibration curve of the electrode, measure the potential and calculate rhe difference of potential
!:1E between the measured potential and ET . !:1E is related to
the following holds: E = Eb + S log C¡ the concentration of the ion to be detertnined by the
and for: - log C¡ == pC¡ : E == E I O - SpC¡ equation:
The potentiometric detertnination of the ion concentration is

GsVs)
carried out by measuring the potential difference between /::"E = Slog ( 1 + GTVT
two suitable electro des immersed in the solution to be
examinedi the indicator electrode is selective for the ion to be
or
detertnined and the other is a reference electrode.
Apparatus Use a voltmeter allowing measurements to the !:lE GsVs
nearest 0.1 millivolt and whose input impedance is at least lOS = 1 + GTVT
one hundred times greater than that of the electrodes used.
V-A270 Appendix VIII F 2014
volume of the test solution, the same manner as solution (2) but adding sufficient of the
concentration of the ion to be deterrnined in the test internal standard to produce a final concentration of
solution, 5.0 % v/v.
Vs added volume of the reference solution, The chromatographic pro ce dure may be carried out using a
Cs concentration of the ion to be deterrnined in the column (l.5 m x 4 mm) packed with porous polymer beads
reference solution, (100 to 120 mesh) (Porapak Q and Chromosorb 101 are
s slope of the electro de deterrnined experimentally, at suitable) and rnaintained at 150° with both the inlet port and
constant temperature, by measuring the difference the detector at 170°.
between the potentials obtained with two reference Calculate the percentage content of ethanol frorn the areas of
solutions whose concentrations differ by a factor of the peaks due to ethanol in the chromatograrns obtained with
ten and are situated within the range where the solutions (1) and (3) .
calibration curve is linear.
Method 11
Plot on a graph 10 I1f (y-axis) against Vs (x-axis) and (No Ph. Eur. method)
extrapolate the line obtained until it intersects the x-axis. For preparations in which, in accordance with the authority
At the intersection, the concentration CT of the test solution given in the monographs, Industrial Methylated Spirit has
in the ion to be determined is given by the equation: been used, determine the content of ethanol as described in
Method I but using as solution (2) a volume of the
preparation being examined diluted with water to contain
between 4.0 and 6.0% v/v of total ethanol and methanol.
Determine the concentration of methanol in the following
Method 111 (single standard addition) manner. Carry out the chromatographic procedure described
under Method I but using the following solutions. Solution
To a volume VT of the test solution prepared as prescribed (1) contains 0.25% v/v of methanol and 0.25% v/v of propan-
in the monograph, add a volume Vs of a reference solution 1-01 (internal standard). For solution (2) dilute a volume of
containing an amount of the ion to be determined known to the preparation being examined with water to contain
give a response situated in the linear part of the calibration berween 0.2 % and 0.3% v/v of methano!. Prepare solution
curve. Prepare a blank solution in the same conditions. (3) in the same manner as solution (2) but adding sufficient
Measure at least three times the potentials of the test solution of the internal standard to produce a final concentration of
and the blank solution, before and after adding the reference 0.25% v/v.
solution. Calculate the concentration CT of the ion to be
The sum of the contents of ethanol and methanol is within
analysed using the following equation and making the
the range specified in the monograph and the ratio of the
necessary corrections for the blank:
content of methanol to that of ethanol is commensurate with
CsVs Industrial Methylated Spirit having been used.
CT = --A~'E'-------------
lOs (VT + Vs) - VT Method III
VT volume of the test solution or the blank, These methods are intended for the examination of liquid
CT concentration of the ion to be deterrnined in the test pharrnaceutical preparations and their ingredients that
solution, contain ethano!. The ethanol content of a liquid is expressed
Vs added volume of the reference solution, as the number of volumes of ethanol contained in
Cs concentration.of the ion to be deterrnined in the 100 volumes of the liquid, the volumes being measured at
reference solution, 20 ± 0.1 oc. This is known as the 'percentage of ethanol by
!lE difference berween the average potentials measured volume' (per cent V/V). The content may also be expressed
before and after adding Vs, in grams of ethanol per 100 g of the liquido This is known as
S slope of the electrode deterrnined experimentally, at the 'percentage of ethanol by mass' (per cent m /m).
constant temperature, by measuring the difference
between the potentials obtained from two reference MethodA
solutions whose concentrations differ by a factor of Where preparations contain dissolved substances, the
ten and are situated within the range where the dissolved substances must be separated from the ethanol that
calibration curve is linear. is to be deterrnined by distillation. Where distillation would
distil volatile substances other than ethanol and water, the
appropriate precautions are stated in the monograph.
The relation berween the density at 20 ± 0.1 oC, the relative
F. Determination of Ethanol density (corrected to vacuum) and the ethanol content of a
Use Method I or, where appropriate, Method II unless mixture of water and ethanol is given in the tables of the
otherwise prescribed in the monograph. Internatíonal Organisation for Legal Metrology (1972),
International Recommendation No . 22.
Method I
Carry out the method for gas chromatography,
Appendix III B, using the following solutions. Solution (1)
contains 5.0% v/v of absolute ethanol and 5.0% v/v of propan-
1-01 (internal standard). For solution (2) dilute a volume of
the preparation being examined with water to contain
between 4.0 and 6.0% v/v of ethanol. Prepare solution (3) in
2014 Appendix VIII F V-A271
Hydrometer method Transfer 50.0 mL of the

preparation to be examined, measured at 20 ± 0.1 oC, to
the distillation fiask, add 200-300 mL of distilled water R and
distil, as described above, into a volumetric fiask until at
least 180 mL has been collected. Adjust the temperature to
20 ± 0.1 oC and dilute to 250.0 mL with distilled water R at
20 ± 0.1 oC.
Transfer the distillate to a cylinder whose diameter is at least
6 mm wider than the bulb of the hydrometer. If the volume
is insufficient, double the quantity of the sample and dilute
the distillate to 500.0 mL with distilled water R at
A 20 ± 0.1 oc.
Multiply the strength by 5 10 allow for the dilution during
+--- c the determination. After calculation of the ethanol content
using Table 2.9. 10.-1 , round offthe result to 1 decimal
place.
MethodB
Head-space gas chromatography (2.2.28).
Internal standard solution Dilute 1.0 mL of propanol Rl
to 100.0 mL with water R. Dilute 1.0 mL of the solution 10
--'-- -- o
Test solution Dilute a volume of the preparation to be
examined corresponding 10 1 g of ethanol to 50.0 mL with
water R . Dilute 1.0 mL of the solution 10 20.0 mL with
water R . To 2.0 mL of this solution add 1.0 mL of the
intemal standard solution and dilute to 20.0 mL with
water R.
Referenee solution (a) Dilute 5.0 mL of anhydrous
E ethanol R to 100.0 mL with water R. Dilute 25.0 mL of the
solution to 100.0 mL with water R. Dilute l.0 mL of this
solution to 20.0 mL with water R .
Referenee solution (b) Mix 0.5 mL of reference solution
(a) and 1.0 mL of the intemal standard solution and dilute
Figure 2.9.10.·1. - Apparatus (ar the determinatian a( ethanal to 20.0 mL with water R .
cantent Referenee solution (e) Mix 1.0 mL of reference solution
Dimensians in millimetres (a) and 1.0 mL of the intemal standard solution and dilute
Apparatus The apparatus (see Figure 2.9.10.-1) consists Referenee solution (d) Mix l.5 mL of reference solution
of a round-bottomed fiask (A) fitted with a distillation head (a) and 1.0 mL of the intemal standard solution and dilute
(E) with a steam trap and attached to a vertical condenser 10 20 .0 mL with water R.
(C). The latter is fitted at itslower part with a tube (D), Referenee solution (e) Dilute 1.0 mL of methanol R2 to
which carries the distillate into the lower part of a 100 mL 100.0 mL with water R. Dilute l.0 mL of the solution to
or 250 mL volumetric fiask. The volumetric fiask is 20.0 mL with water R .
irnmersed in a mixture of ice and water (E) during the Referenee solution (f) Mix 1.0 mL of the intemal
distillation. A disc having a circula" aperture 6 cm in standard solution, 2.0 mL of reference solution (a) and
diameter is placed under the fiask (A) to reduce the risk of 2.0 mL of reference solution (e) and dilute to 20.0 mL with
charring of any dissolved substances. water R.
METHOD Column:
Pyenometer methodJoseillating transdueer density - material: fused silica;
meter method Transfer 25.0 mL of the preparation to be
- size: 1 = 30 m, 0 = 0.53 mm;
examined, measured at 20 ± 0.1 oC, to the distillation fiask.
Dilute with 100-150 mL of distilled water R and add a few - stationary phase:
pieces of pumice. Attach the distillation head and condenser. poly[(cyanopropyl) (phenyl)J[dimethyl]siloxane R (film
Distil and collect not less than 90 mL of distillate in a thickness 3 ¡.tm).
100 mL volumetric fiask. Adjust the temperature to Carrier gas helium for chromatography R .
20 ± 0.1 oC and dilute to 100.0 mL with distilled water R at Flow rate 3 mUmin.
20 ± 0.1 oc. Determine the relative density at 20 ± 0.1 oC Split ratio 1:50.
using a pycnometer or an oscillating transducer density
Static head-space conditions that may be used:
meter.
- equilibration temperature: 85 oC;
The values indicated in Table 2.9.10.-1, column 3, are
multiplied by 4 to obtain the percentage of ethanol by - equilibration time: 20 mino
volume (V/V) contained in the preparation. After calculation
of the ethanol content using the table, round off the result to
1 decimal place.
V-A272 Appendix VIII F 2014
Temperature: P,. Relative density of the Ethanol content in

(kg'III- ') distillate measured in air per cent V/V
Time Temperature 20
d 20 at 20 oC
(min) (OC)
0·1.6 40 983.5 0.9853 11.02
Colurnn
1.6·9.9 40 -765 984.0 0.9858 10.60
9.9·13.6 65 -7 175 984.5 0.9863 10.18
13.6·20 175 985.0 0.9868 9.76
Injection port 200 985.5 0.9873 9.35
Detector 200 986.0 0.9878 8.94
986.5 0.9883 8.53

987.0 0.9888 8.13
Injeetion 1.0 mL of the gaseous phase of the test solution
and referenee solutions (b), (e), (d) and (f), at least 3 times. 987.5 0.9893 7.73
Elution order Methanol, ethanol, l-propano!. 988.0 0.9898 7.34

Relative retention With referenee ro ethanol (retention 988.5 0.9903 6.95
time = about 5.3 min) : methanol = about 0.8;
989.0 0.9908 6.56
1-propanol = about 1.6.
989.5 0.9913 6.17
Table 2.9.10.·1. - Relatianship between density, relative
density and ethanal cantent 990.0 0.9918 5.79
P,. Relative densit)' of the Ethanol content in 990.5 0.9923 5.42
(kg'm-') distillate measured in air per cent V/V
991.0 0.9928 5.04
d 20
20 at 20 oC
968.0 0.9697 991.5 0.9933 4.67
25.09
968.5 0.9702 24.64 992.0 0.9938 4.30
969.0 0.9707 24.19 992.5 0.9943 3.94
969.5 0.9712 23.74 993.0 0.9948 3.58
970.0 0.9717 23.29 993.5 0.9953 3.22
970.5 0.9722 22.83 994.0 0.9958 2.86
971.0 0.9727 22.37 994.5 0.9963 2.51
971.5 0.9733 21.91 995.0 0.9968 2.16
972.0 0.9738 21.45 995.5 0.9973 1.82
972.5 0.9743 20.98 996.0 0.9978 1.47
973.0 0.9748 20.52 996.5 0.9983 1.13
973.5 0.9753 20.05 997.0 0.9988 0.80
974.0 0.9758 19.59 997.5 0.9993 0.46
974.5 0.9763 19.12 998.0 0.9998 0.13
975.0 0.9768 18.66
975.5 0.9773 18.19 System suitability Referenee solution (f):

976.0 0.9778 17.73
- reso/ution: minimum 5 between the peaks due to methanol
and ethano!.
976.5 0.9783 17.25
Establish a ealibration curve with the eoneentration of
977.0 0.9788 16.80 ethanol in referenee solutions (b), (e), (d) and (f) as the
977.5 0.9793 16.34 abseissa and the mean ratio of the peak area of ethanol ro the
peak area of the intemal standard in the eorresponding
978.0 0.9798 15.88
ehromarograms as the ordinate.
978.5 0.9803 15.43 Calculate the pereentage eontent of ethanol in the
979.0 0.9808 14.97 preparation ro be examined.
979.5 0.9813 14.52 Method e
980.0 0.9818 14.07 Gas ehromarography (2.2.28).
980.5 0.9823
Internal standard solution Dilute 1.0 mL of propanol RI
13.63
981.0 0.9828 13.18
Test solution Dilute a volume of the preparation ro be
981.5 0.9833 12.74 examined eorresponding ro 1 g of ethanol to 50.0 mL with
982.0 0.9838 12.31 water R. To 1.0 mL of this solution add 1.0 mL of the
intemal standard solution and dilute to 20.0 mL with
982.5 0.9843 11.87 water R.
983.0 0.9848 11.44
2014 Appendix VIII G V-A273
Referenee solution (a) Dilute 1.0 mL of anhydrous

ethanol R 10 50.0 mL with water R.
G. Determination of Methanol and
Referenee solution (b) Dilute 1.0 mL of methanol R2 10 Propan-2-ol
100.0 mL with water R. Dilute 1.0 mL of the solution to (Ph. Eur. method 2.9.11)
20 .0 mL with water R.
MethodA
Referenee solution (e) Mix 1.0 mL of the internal
standard solution, 1.0 mL of referenee solution (a) and Head-spaee gas ehromatography (2.2.28).
2.0 mL of referenee solution (b) and dilute 10 20.0 mL with Internal standard solution Dilute 1.0 mL of propanol R1
water R . to 100.0 mL with water R. Dilute 1.0 mL of the solution 10
Column: 20 .0 mL with water R.
- material: fused siliea; Test solution Mix 1.0 mL of the internal standard
solution and 4.0 mL of the preparation to be examined and
- size: 1 = 30 m, 0 = 0.53 mm; dilute 10 20.0 mL with water R.
- stationary phase:
Referenee solution (a) Mix 1.0 mL of methanol R2 and
poly[(cyanopropyl) (phenyl) J[dimethyl]siloxane R (film
1.0 mL of 2-propanol R2 and dilute to 100.0 mL with
thiekness 3 ~lm). water R . Dilute 1.0 mL of the solution to 20.0 mL with
Carrier gas helium for chromatography R. water R.
Flow rate 3 mUmin. Referenee solution (b) Dilute 5.0 mL of anhydrous
Split ratio 1:50. ethanol R to 100.0 mL with water R. Dilute 25.0 mL of the
Temperature: solution to 100.0 mL with water R. Dilute 1.0 mL of this
solution to 20 .0 mL with water R.
Time Temperature
(min) (oC) standard solution, 2.0 mL of referenee solution (a) and
CoJumn 0·1.6 40 2.0 mL of referenee solution (b) and dilute to 20.0 mL with
water R .
1.6 - 9.9 40 ..... 65
Column:
9.9·13.6 65 ..... 175
- material: fused siliea;
13.6 - 20 175 - size: 1 = 30 m, 0 = 0.53 mm;
lnjection port 200 - stationary phase:
Detector 200 poly[(cyanopropyl) (phenyl)J[dimethyl]szloxane R (film
thickness 3 ¡.tm).
Carrier gas helium for chromatography R.
Deteetion Flame ionisation.
Flow rate 3 mUmin.
Injeetion 1.0 ¡.tL of the test solution and referenee solution
Split ratio 1:50.
(e), at least 3 times.
Elution order Methanol, ethanol, 1-propano!.
Relative retention With referenee 10 ethanol (retention
time = about 5.3 min): methanol = about 0.8; - equilibration time: 20 mino
1-propanol = about 1.6. Temperature:
System suitability Referenee solution (e) :
- resolution: minimum 5 between the peaks due to methanol Time Temperature
(min) (OC)
and ethano!.
CoJumn 0-1.6 40
Calculate the ethanol eontent in per eent V/V using the
following expression: 1.6 - 9.9 40 ..... 65
9.9 - 13.6 65 ..... 175

Al X h x 100 13.6 - 20 175
A 2 X h X VI
Injection port 200
Detector 200
area of the peak due 10 ethanol in the ehroma1Ogram
obtained with the test solution;
area of the peak due 10 ethanol in the ehromatogram Deteetion Flame ionisation.
obtained with referenee solution (e); Injeetion: 1.0 mL of the gaseous phase of the test solution
11 are a of the peak due 10 the internal standard in the and referenee solution (e), at least 3 times.
ehroma1Ogram obtained with the test solution;
Elution order Methanol, ethanol, 2-propanol, 1-propano!.
area of the peak due 10 the internal standard in the
ehromatogram obtained with referenee solution (e); Relative retention With referenee 10 ethanol (retention
volume of the preparation to be examined in the test time = about 5.3 min): methanol = about 0.8;
solution, in millilitres. 2-propanol = about 1.2; 1-propanol = about 1.6.
System suitability Referenee solution (e):
- resolution: minimum 5 between the peaks due to methanol
and ethano!.
Calculate the methanol eoment in per eem V/V using the
following express ion:
V-A274 Appendix VIII H 2014

Injeetion 1.0 JlL of the test solution and referenee solution
Ce), at least 3 times .
area of the peak due to methanol in the
Elution order Methanol, ethanol, 2-propanol, 1-propano!.
ehromatogram obtained with the test solution;
area of the peak due to methanol in the Relative retention With referenee to ethanol Cretention
ehromatogram obtained with referenee solution Ce); time = about 5.3 min): methanol = about 0.8;
area of the peak due to the internal standard in the 2-propanol = about 1.2; 1-propanol = about 1.6.
ehromatogram obtained with the test solution; System suitability Referenee solution Ce) :
are a of the peak due to the internal standard in the - resolution: minimum 5 between the peaks due to methanol
ehromatogram obtained with referenee solution Ce) . and ethanol.
Calculate the 2-propanol eontent in per eent VN using the Calculate the methanol eontent in per cent V/V using the
following expression: following expression:
area of the peak due to 2-propanol in the area of the peak due to methanol in the
ehromatogram obtained with the test solution; ehrornatogram obtained with the test solution;
area of the peak due to 2-propanol in the area of the peak due to methanol in the
ehromatogram obtained with referenee solution Ce); ehromatogram obtained with referenee solution Ce);
area of the peak due to the internal standard in the area of the peak due to the internal standard in the
ehromatogram obtained with the test solution; ehromatogram obtained with the test solution;
area of the peak due to the internal standard in the area of the peak due to the internal standard in the
ehromatogram obtained with referenee solution Ce). ehromatogram obtained with referenee solution Ce).
MethodB Calculate the 2-propanol eontent in per cent VN using the
Gas ehromatography (2.2.28). following expression:
Internal standard solution Dilute 1.0 mL of propanol Rl
to 100.0 mL with water R .
Test solution Mix 1.0 mL of the internal standard
solution and 4.0 mL of the preparation to be examined and area of the peak due to 2-propanol in the
dilute to 20.0 mL with water R. ehromatogram obtained with the test solution;
Referenee solution (a) Mix 1.O mL of methanol R2 and area of the peak due to 2-propanol in the
1.0 mL of 2-propanol R2 and dilute to 100.0 mL with ehromatogram obtained with referenee solution Ce);
water R . Dilute 1.0 mL of the solution to 20.0 mL with area of the peak due to the internal standard in the
water R. chromatogram obtained with the test solution;
Referenee solution (b) Dilute 1.0 mL of anhydrous area of the peak due to the internal standard in the
ethanol R to 50.0 mL with water R . chromatogram obtained with referenee solution Ce).
standard solution, 1.0 mL of referenee solution Cb) and
2.0 mL of referenee solution Ca) and dilute to 20.0 mL with
water R .
H. Determination of Nitrogen
U se Method I unless otherwise speeified in the monograph.
Column:
- material: fused siliea; Method 1
- size: l = 30 m, 0 = 0.53 mm; (Ph. Eur. method 2.5.9)
- stationary phase: SEMI-MICRO METHOD
poly[(cyanopropyl) (phenyl)][dimethyl] siloxane R Cfilm Place a quantity of the substanee to be examined Cm g)
thiekness 3 Jlm). eontaining about 2 mg of nitrogen in a eombustion f1ask, add
Carrier gas helium for chromatography R. 4 g of a powdered mixture of 100 g of dipotassium sulfate R,
Flow rate 3 mUmin. 5 g of copper sulfate R and 2.5 g of selenium R, and three glass
beads. Wash any adhering particles from the neek into the
Split ratio 1:5 0.
f1ask with 5 mL of sulfuric acid R, allowing it to run down the
Temperatul'e: sides of the f1ask, and mix the eontents by rotation . Close the
mouth of the f1ask loosely, for example by means of a glass
Time Temperalure bulb with a short stem, to avoid excessive loss of sulfurie
(min) (oC)
aeid. Heat gradually at first, then inerease the temperature
Column 0·1.6 40
until there is vigorous boiling with condensation of sulfurie
1.6· 9.9 40 -; 65 aeid in the neek of the f1ask; preeautions should be taken to
9.9 ·13.6 65 -; 175
prevent the upper part of the f1ask from becoming
overheated. Continue the heating for 30 min, unless
13.6·20 175 otherwise preseribed. Cool, dissolve the solid material by
Injection port 200 eautiously adding to the mixture 25 mL of water R, eool
again and place in a steam-distillation apparatus . Add 30 mL
Detector 200
of strong sodium hydroxide solution R and distil immediately by
2014 Appendix VIII K V-A275
passing steam through the mixture. Collect about 40 mL of Carry out the following procedure protected from light.
distillate in 20.0 mL of 0.01 M hydrochloric acid and enough Dissolve a quantity of the substance being examined in
water R ro cover the tip of the condenser. Towards the end sufficient aldehyde-free absolute ethanol to produce a solution
of the distillation, lower the receiver so that the tip of the containing 300 to 350 ¡lg in 10 mL unless otherwise
condenser is aboye the surface of the acid. Take precautions specified in the monograph. Transfer 10 mL to a 25-mL
to prevent any water on the outer surface of the condenser graduated fiask, add 2 mL of triphenyltetrazoliUll1 chloride
from reaching the contents of the receiver. Titrate the solution, displace the air in the fiask with oxygen-free nitrogen,
distillate with 0.01 M sodium hydroxide, using methyl red mixed immediately add 2 mL of dilute tetramerhylammoniwn
sofurion R as indicaror (nI mL of 0.01 M sodium hydroxide). hydroxide solution and again displace the air with oxygen-ji·ee
Repeat the test using about 50 mg of gfucose R in place of the nitrogen. Stopper the fiask, mix the contents by gently
substance to be examined (n2 mL of 0.01 M sodium swirling and allow to stand in a water bath at 30° for 1 hour
hydroxide). unless otherwise specified in the monograph. Cool rapidly,
add sufficient aldehyde-free absolure ethanol ro produce 25 mL,
mix and immediately determine the absorbance of the
Content of nitro gen = 0.01401 m(n2 - n¡)
per cent resulting solution in a slOppered cell at the maximum at
485 nm, Appendix II B, using in the reference cell a solution
prepared at the same time and in the same manner using
10 mL of aldehyde-free absolute ethanol. Repeat the operation
Method 11 (Detennination of Protein in Blood
Products) using the specified BPCRS or EPCRS in place of the
substance being examined ensuring that the period of time
(No Ph. Eur. method) that elapses between the addition of the
For dried blood products prepare a solution of the tetramethylammonium hydroxide solution and the
preparation as directed in the monograph. measurement of the absorbance is the same as for the test
To a volume expected ro contain about 0.1 g of protein add solution.
sufficient saline solwion to produce 20 mL. To 2 mL of the
resulting solution, in a 75-rnL boiling tube, add 2 mL of a
solution containing 75% v/v of nitrogen-free sulfuric acid,
4.5% w/v of potassium sulfate and 0.5% w/v of copper(Il) K. Ethylene Glycol and Diethylene Glycol
sulfate, mix and loosely stopper the tube. Reat gradually ro
boiling, boil vigorously for 1.5 hours and coo!. If the solution in Ethoxylated Substances
is not clear add 0.25 mL of hydrogen peroxide solurion (Ph. Eur. method 2.4.30)
(20 vol), continue heating until a clear solution is produced Ethoxylated substances may contain varied amounts of ethylene
and coo!. During heating, take precautions ro ensure that the glycol and diethylene glycol, as a result of the manufacuuing
upper pan of the tube is not overheated. process. The follo wing method may be used for the quantitative
Transfer the solution to a distillation apparatus using three determination of these substances, in particular in the case of the
3-mL quantities of water, add 10 mL of 10M sodium hydroxide following surfactants: macrogolglycerol ricino/eate, macrogo/glycero/
and distil rapidly for 4 minutes, collecting the distillate in a hydroxystearate, macrogol 15 hydroxystearate, nonoxinol 9 and
mixture of 5 mL of a saturated solution of boric acid and macrogol celOstearyl ether.
5 mL of water and keeping the tip of the condenser below Gas chromalOgraphy (2.2.28).
the level of the acid. Lower the collection fiask so that the Internal standard solution Dissolve 30.0 mg of
condenser can drain freely and continue the distillation for a 1,2-pentanediol R in acelOne R and dilute to 30.0 rnL with the
further 1 minute. Titrate with 0.02M hydrochloric acid VS same solvento Dilute 1.0 mL of this solution lO 20.0 mL
using methyl red mixed solurion as indicaror (VI mL). with acetone R.
To a further volume of the preparation being examined, or of Test solution Dissolve 0.500 g of the substance to be
the solution prepared from it, expected to contain about examined in the internal standard solution and dilute to
0.1 g of protein, add 12 mL of satine solurion, 2 mL of a 10.0 mL with the same solution.
7.5% w/v solution of sodium molybdate and 2 mL of a mixture
Reference solution (a) Mix 30.0 mg of ethy lene glycol R
of 1 volume of nitrogen-free sulfuric acid and 30 volumes of
with acelOne R and dilute ro 100.0 mL with the same
water. Shake, allow ro stand for 15 minutes, add sufficient
solvento Dilute 1.0 mL to 10.0 mL with the internal
water ro produce 20 mL, shake again and centrifuge. Using
standard solution.
2 mL of the resulting clear supernatant liquid repeat the
procedure described aboye beginning at the words 'in a Reference solution (b) Prepare a solution of diethy lene
75-rnL boiling tube .. . ' (V2 mL). Calculate the protein glycol R with a concentration corresponding to the prescribed
content in mg per mL of the preparation being examined, limit and using the same solvents as for the preparation of
using the expression 6.25 x 0.280(V]-V2 ) and taking into reference solution (a).
account the initial dilution. Column:
- material: fused silica,
- size: l = 30 m, 0 = 0.53 mm,
- stationary phase: macrogol 20 000 R (film thickness 1 ~]m) .
J. Tetrazolium Assay of Steroids Carrier gas heliUln for chromalOgraphy R .
Flow rate 10 mUmin.
The caloured reaaion products tend 10 adsorb onlO the sUlface of
the glassware. To avoid low results, the glassware should be treated Split ratio 1:3 .
with the coloured reaaion produas before use. The treated
glassware should be reserved for this assay and should be washed
only with water between assays.
V-A276 Appendix VIII L 2014
Tetnperature: Sample solution (2) Dissolve 0.200 g of the substance to be

examined in N,N-dimethylformamide R (DMF) and dilute ro
20.0 mL with the same solvent.
Time Temperature
(mio) (OC) Sample prepararlon 3 This is intended for the control of
Column 0· 40 80 ~ 200 N,N-dimethylacetamide andlor N,N-dimethylformamide,
when it is known or suspected that one or both of these
40·45 200 ~ 230
substances are present in the substance ro be examined.
45·65 230
Sample solution (3) Dissolve 0.200 g of the substance to be
Injection port 250 examined in 1,3-dimethy l-2-imidaz olidinone R (DMI) and
Detector 250 dilute to 20.0 mL with the same solvent.
In sorne cases none of the aboye sample preparation
procedures are appropriate, in which case the diluent ro be
Detection Flame ionisation. used for the preparation of the sample solution and the static
Injection 2 ¡.¡L. head-space conditions to be employed must be demonstrated
Relative retention With reference ro 1,2-pentanediol to be suitable.
(retention time = about 19 min): ethylene glycol = about Solvent solution (a) To 1.0 mL of Glass 1 residual solvent
0.7; diethylene glycol = about 1.3. solution GRS, add 9 mL of dimethyl sulfoxide R and dilute ro
100.0 mL with water R. Dilute 1.0 mL of this solution to
100 mL with water R. Dilute 1.0 mL of this solution ro
L Residual Solvents The reference solutions correspond ro the following limits:
(Ph. Eur. method 2.4.24) - benzene: 2 ppm,
The test pro ce dures described in this general method may be - carbon tetrachloride: 4 ppm,
used: - 1,2-dichloroethane: 5 ppm,
i. for the identification of the majority of Class 1 and Class 2 - 1,1-dichloroethene: 8 ppm,
residual solvents in an active substance, excipient or - 1,1,1-trichloroethane: 10 ppm .
medicinal product when the residual solvents are unknoWll;
Solvent solution (b) Dissolve appropriate quantities of the
ii. as a limit test for Class 1 and Class 2 solvents when Class 2 residual solvents in dimethyl sulfoxide R and dilute ro
present in an active substance, excipient or medicinal 100.0 mL with water R. Dilute to give a concentration of
product;
1120 of the limits stated in Table 2 (see 5.4. R esidual
iii. for the quantification of Class 2 solvents when the limits solvents).
are greater than 1000 ppm (0.1 per cent) or for the Solvent solution (e) Dissolve 1.00 g of the solvent or solvents
quantification of Class 3 solvents when required. present in the substance to be examined in dimethyl
Class 1, Class 2 and Class 3 residual solvents are listed in sulfoxide R or water R, if appropriate, and dilute to 100.0 mL
general chapter 5. 4. Residual solvents. with water R. Dilute ro give a concentration of 1120 of the
Three diluents are described for sample preparation and the limit(s) stated in Table 1 or 2 (see 5.4. Residual solvents).
conditions to be applied for head-space injection of the Blank solution Prepare as described for solvent solution (c)
gaseous sample onto the chromatographic system. but without the addition of solvent(s) (used to verify the
Two chromatographic systems are prescribed but System A is absence of interfering peaks).
preferred whilst System B is employed normally for Test solut¡on Introduce 5.0 mL of the sample solution and
confirmation of identity. The choice of sample preparation 1.0 mL of the blank solution into an injection vial.
procedure depends on the solubility of the substance ro be
Referenee solution (a) (Glass 1) Introduce 1.0 mL of solvent
examined and in certain cases the residual solvents ro be
controlled. solution (a) and 5.0 mL of the appropriate diluent into an
injection vial.
The following residual solvents are not readily detected by
Referenee solution (aJ (Glass 1) Introduce 5.0 mL of the
the head-space injection conditions described: formamide,
2-ethoxyethanol, 2-methoxyethanol, ethylene glycol, sample solution and 1.0 mL of solvent solution (a) into an
injection vial.
N-methylpyrrolidone and sulfolane . Other appropriate
procedures should be employed for the control of these R eference solution (b) (Glass 2) Introduce 1.0 mL of solvent
residual solvents. solution (b) and 5.0 mL of the appropriate diluent into an
When the test procedure is applied quantitatively ro control injection vial.
residual solvents in a substance, then it must be validated. Referenee solution (e) Introduce 5.0 mL of the sample solution
and 1.0 mL of solvent solution (c) into an injection vial.
PROCEDURE R eferenee solution (d) Introduce 1.0 mL of the blank solution
Examine by gas chromatography with static head-space and 5.0 mL of the appropriate diluent into an injection vial.
injection (2.2.28). Glose the vials with a tight rubber membrane stopper eoated with
Sample prepararlon 1 This is intended for the control of polytetrafiuoroethylene and seeure with an aluminium erimped
residual solvents in water-soluble substances. cap. Shake to obtain a homogeneous solution.
Sample solution (1) Dissolve 0.200 g of the substance ro be The following sta tic head-space injection conditions may be
examined in water R and dilute to 20 .0 mL with the same used:
solvent.
Sample prepararlon 2 This is intended for the control of
residual solvents in water-insoluble substances.
2014 Appendix VIII L V-A277
Sample preparatioo Inject 1 mL of the gaseous phase of the test solution onto the
procedure column described in System A. If in the chromatogram
Operatiog parameters 2 3 obtained, there is no peak which corresponds to one of the
EquiJibration temperature (OC) 80 105 80 residual solvent peaks in the chromatograms obtained with
reference solution (a) or (b), then the substance to be
EquiJibration time (min) 60 45 45
examined meets the requirements of the test. If any peak in
Transfer-lioe temperature (oC) 85 110 105 the chromatogram obtained with the test solution
corresponds to any of the residual solvent peaks obtained
Carrier gas: Nitrogen for chromatography R or Helium for
chromatographv R at an appropriate pressure with reference solution (a) or (b) then System B is to be
Pressurisation time (s) 30 30 30 employed.
Injeetion volume (mL) Inject 1 mL of the gaseous phase of referenee solution (a)
onto the column described in System B and record the
chromatogram under such conditions that the signal-to-noise
The chromatographic procedure may be carried out using: ratio for benzene can be measured. The signal-to-noise ratio
SystemA must be at least 5. A typical ehromatogram is shown in
- a fused-silica capillary or wide-bore column 30 m long Figure 2.4.24.-3.
and 0.32 mm or 0.53 mm in intemal diameter coated Inject 1 mL of the gaseous phase of reference solution (al)
with cross-linked 6 per cent onto the column described in System B. The peaks due to
polycyanopropylphenylsiloxane and 94 per cent the Class 1 residual solvents are still detectable.
polydimethylsiloxane (film thickness: 1.8 ~m or 3 ~lm), Inject 1 mL of the gaseous phase of referenee solution (b)
- nitrogen for chromatography R or heliwn for onto the column described in System B and record the
chromatography R as the carrier gas, split ratio 1:5 with a chromatogram under such eonditions that the resolution
linear velociry of about 35 cm/s, between aeetonitrile and trichloroethene can be deterrnined.
- a flame-ionisation detector (a mas s spectrometer may also The system is suitable if the chromatogram obtained
be used or an electron-capture detector for the chlorinated resembles the chromatogram shown in Figure 2.4.24.-4 and
residual solvents of Class 1), the resolution between acetonitrile and trichloroethene is at
least 1.0.
maintaining the temperature of the column at40 oC for
20 min, then raising the temperature at a rat.e of 10 °C per Inject 1 mL of the gaseous phase of the test solution onto the
min to 240 oC and maintaining it at 240 oC for 20 min and column described in System B. If in· the chromatogram
maintaining the temperature of the injection port at 140 oC obtained, there is no peak which corresponds to any of the
and that of the detector at 250 oC, or, where there is residual solvent peaks in the chromatogram obtained with the
interference from the matrix, use: reference solution (a) or (b), then the substance to be
examined meets the requirements of the test. If any peak in
SystemB the chromatogram obtained with the test solution
- a fused-silica capillary or wide-bore column 30 m long corresponds to any of the residual solvent peaks obtained
and 0.32 mm or 0.53 mm in intemal diameter coated with reference solution (a) or (b) and confirrns the
with macrogo! 20 000 R (film thickness: 0.2 5 ~m) , correspondence obtained when using System A, then proceed
- nitrogen for chromatography R or helium for as follows.
chromatography R as the carrier gas, split ratio 1:5 with a Inject 1 mL of the gaseous phase of reference solution (c)
linear velocity of about 35 cm/s. onto the column described for System A or System B.
- a flame-ionisation detector (a mass spectrophotometer If necessary, adjust the sensitivity of the system so that the
may also be used or an electron-capture detector for the height of the peak corresponding to the identified residual
chlorinated residual solvents of Class 1), solventes) is at least 50 per cent of the full scale of the
maintaining the temperature of the column at 50 oC for recorder.
20 min, then raising the temperature at arate of 6 oC per Inject 1 mL of the gaseous phase of reference solution (d)
min to 165 oC and maintaining it at 165 oC for 20 min and onto the column. No interfering peaks should be observed.
maintaining the temperature of the injection port at 140 oC Inject 1 mL of the gaseous phase of the test solution and
and that of the detector at 250 oc. 1 mL of the gaseous phase of reference solution (e) on to the
Inject 1 mL of the gaseous phase of reference solution (a) column. Repeat these injections twice more.
onto the column described in System A and record the The mean area of the peak of the residual solventes) in the
chromatogram under such conditions that the signal-to-noise chromatograms obtained with the test solution is not greater
ratio for 1,1,l-trichloroethane can be measured. The signal- than half the mean area of the peak of the eorresponding
to-noise ratio must be at least 5. A typical chromatogram is residual solventes) in the chromatograms obtained with
shown in Figure 2.4.24.-l. reference solution (c). The test is not valid unless the relative
Inject 1 mL of the gaseous phase of reference solution (al) standard deviation of the differences in areas between the
onto the column described in System A. The peaks due to analyte peaks obtained from 3 replicate paired injections of
the Class 1 residual solvents are still detectable. reference solution (c) and the test solution, is at most
Inject 1 mL of the gaseous phase of reference solution (b) 15 per cent.
onto the column described in System A and record the A flow diagram of the procedure is shown in Figure
chromatogram under such eonditions that the resolution 2.4.24.-5.
between acetonitrile and methylene chloride can be When a residual solvent (Class 2 or Class 3) is present at a
deterrnined. The system is suitable if the chromatogram level of 0.1 per cent or greater then the content may be
obtained resembles the chromatogram shown in Figure quantitatively deterrnined by the method of standard
2.4.24.-2 and the resolution between acetonitrile and additions.
methylene ehloride is at least 1.0.
2 4/5
~---..,JI.._----.....J ---_-J~~
o 2 3 4 5 6 7 8 9 10 11 12 13 14 min
1. 1,1-dichloroethene 2. 1,1,1·trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethane
Figure 2.4.24.-1. - Typical chromafogram of class 1 solvents using the conditions described for Sysfem A and Procedure 1.
Flame-ionisation detector.
3 4 5/6 8 11 14 1617
18
17
10
15 j
~
17
~
7
l
1
A. J '--.!t.,-, 9 vi 1~
13
..1'
o 5 10 15 20 25 30 min
1. methanol 5. cis-1,2-dichloroethene 9. 1,2-dimethoxyethane 13. pyridine 16. chlorobenzene
2. acetonitrile 6. nitromethane 10. 1,l,2-trichloroethene 14. toluene 17. xylene ortho, meta, para
3. dichloromethane 7. chloroform 11. methylcyclohexane 15. 2-hexanone 18. tetralin
4. hexane . 8. cyclohexane 12. l,4-dioxan
Figure 2.4.24.-2. - Chromatogram of Class 2 solvents using the conditions described for Sysfem A and Procedure 1.
Flame-ionisation detector.
2014 Appendix VIII L V-A279
2/3
o 2 3 min
1. 1,l-dichloroethene 2. l.l,l-trichloroethane 3. carbon tetrachloride 4. benzene 5. 1,2-dichloroethane
Figure 2.4.24.-3. - Chromatogram of Class 1 residual solvents using the conditions described for System B and Procedure 1.
Flame-ionisation delector.
if
4811 5/10 14 17
3/9
17
16
IfA
U'- ~l
15 17
U 'v W'-------' 13
, -__~J \'-___________________
o 2 3 4 5 6 7 8 9 min
1. methanol 5. cis-1,2-dichloroethene 9. 1,2-dimethoxyethane 13. pyridine 16. chlorobenzene
2. acetonitrile 6. nitromethane 10. 1,I,2-trichloroethene 14. toluene 17. xylene ortho, meta, para
3. dichloromethane 7. chloroform 11. methylcyclohexane 15. 2-hexanone 18. tetralin (IR = 28 mini
4. hexane 8. cyclohexane 12. 1,4-dioxan
Figure 2.4.24.-4. - Typical chromatogram of class 2 residual solvents using the conditions described for System B and
NO PASSES THE TEST

~-~I NO FURTHER ACTION
__~I
,>-NO PASSES THE TEST
NO FURTHER ACTION
PREPARATION OF TEST AND REFERENCE SOLUTIONS
LESS THAN HALF THE ARRA OF THE

OBTAINED WITH THE REFERENCE S
> - - - - - - - - - - -- . llpASSES TEST 11
FAILSTEST
Figure 2.4.24.·5. - Diagram relating to the identification of residual solvents and the application of limit tests
2014 Appendix VIII M V-A281
coated with a 1.0 ¡lm thick layer of

M. Residual Ethylene Oxide and Dioxan poly(dimethyl)siloxane R,
(Ph. Eur. method 2.4.25) - helium for chromatography R or nitrogen for
The test is intended for the determination of residual chromatography R as the camer gas with a linear velocity
ethylene oxide and dioxan in samples soluble in water or of about 20 cmls and a split ratio of 1:20,
dimethylacetamide. For substances that are insoluble or - a fiame-ionisation detector,
insufficiently soluble in these solvents, the preparation of the
sample solution and the head-space conditions to be maintaining the temperature of the column at 50 oC for
employed are given in the individual monograph. 5 min, then raising the temperature at arate of 5 oC per
minute to 180 oC and then raising the temperature at arate
Examine by head-space gas chromatography (2.2.28). of 30 oC per minute to 230 oC and maintaining at 230 oC
A. For samples soluble in or miscible with water, the for 5 min; maintaining the temperature of the injection port
following procedure may be used. at 150 oC and that of the detector at 250 oC.
Test solution Weigh 1.00 g (M T ) of the substance to lnject a suitable volume, for example 1.0 mL, of the gaseous
be examined in a 10 mL vial (other sizes may be used phase ofreference solution (b) . Adjust the sensitivity ofthe
depending on the operating conditions) and add 1.0 mL system so that the heights of the peaks due to ethylene oxide
of water R. Close and mix to obtain a homogeneous and acetaldehyde in the chromatogram obtained are at least
solution. Allow to stand at 70 oC for 45 mino 15 per cent of the full scale of the recorder. The test is not
Reference solution (a) Weigh 1.00 g (M R ) of the valid unless the resolution between the peaks corresponding
substance to be examined into an identical 10 mL vial, to acetaldehyde and ethylene oxide is at least 2.0 and the
add 0.50 mL of ethylene oxide solunon R3 and 0.50 mL peak of dioxan is detected with a signal-to-noise ratio of at
of dioxan solution Rl. Close and mix to obtain a least 5.
homogeneous solution. Allow to stand at 70 oC for lnject separately suitable volumes, for example 1.0 mL (or
45 mino the same volume used for reference solution (b)), of the
Reference solution (b) To 0.50 mL of ethylene oxide gaseous phases of the test solution and reference solution (a).
solution R3 in a 10 mL vial add 0.1 mL of a freshly Repeat the procedure twice more.
prepared 10 mglL solution of acetaldehyde R and 0.1 mL Verification of precision
of dioxan solution Rl . Close and mix to obtain a For each pair of injections, calcula te for ethylene oxide and
homogeneous solution. Allow to stand at 70 oC for for dioxan the difference in area between the peaks obtained
45 mino with the test solution and reference solution (a). The test is
B. For samples soluble in or miscible with not valid unless the relative standard deviation of the 3 values
dimethylacetamide, the following procedure may be obtained for ethylene oxide is not greater than 15 per cent
used. and the relative standard deviation of the 3 values obtained
Test solution Weigh 1.00 g (M T ) of the substance to for dioxan is not greater than 10 per cent. If the weighings
be examined in a 10 mL vial (other sizes may be used used for the test solution and reference solution differ from
depending on the operating conditions) and add 1.0 mL 1.00 g by more than 0.5 per cent, the appropriate corrections
of dimethylacetamide R and 0.20 mL of water R. Close must be made.
and mix to obtain a homogeneous solution. Allow to The content of ethylene oxide or dioxan in parts per million
stand at 90 oC for 45 mino is calculated from the expressions:
Reference solution (a) Weigh 1.00 g (MR ) of the
substance to be examined into a 10 mL vial, add AT xC
1. O mL of dimethylacetamide R, 0.10 mL of dioxan
solution R and 0.10 mL of ethylene oxide solution R2.
Close and mix to obtain a homogeneous solution. Allow
AT area of the peak corresponding to ethylene oxide in
to stand at 90 oC for 45 mino
the chromatogram obtained with the test solution,
Reference solution (b) To 0.10 mL of ethylene oxide AR area of the peak corresponding to ethylene oxide in
solution R2 in a 10 mL vial, add 0.1 mL of a freshly the chromatogram obtained with reference solution
prepared 10 mgIL solution of acetaldehyde R and (a),
0.10 mL of dioxan solution R. Close and mix to obtain a MT mass of the substance to be examined in the test
homogeneous solution. Allow to stand at 70 oC for solution, in grams,
45min. MR mass of the substance to be examined in the
The following static head-space injection conditions may be reference solution, in grams,
used: e the amount of ethylene oxide added to reference
- equilibration temperature: 70 oC (90 oC for solutions in solution (a), in micrograms.
dimethylacetamide) ,
- equilibration time: 45 min, DT xC
- transfer-line temperature: 75 oC (150 oC for solutions in (D R x MT) - (DT x MR)
dimethylacetamide),
- camer gas: helium for chromatography R, area of the peak corresponding to dioxan in the
- pressurisation time: 1 min, chromatogram obtained with the test solution,
- injection time: 12 s. area of the peak corresponding to dioxan in the
chromatogram obtained with reference solution (a),
The chromatographic procedure may be carried out using:
e the amount of dioxan added to reference solution (a)
- a capillary glass or quartz column 30 m long and in micrograms.
0.32 mm in internal diameter the inner surface of which is
V -A282 Appendix VIII N 2014
to dissolve and dilute to 50.0 mL with water R. Dilute

N. N,ALDimethylaniline 5.0 mL of this solution to 250.0 mL with water R.
(Ph. Eur. method 2.4.26) To 1.0 mL of the latter solution in a ground-glass-stoppered
tube add 5 mL of 1 M sodium hydroxide and 1.0 mL of the
MethodA internal standard solution. Stopper the tube and shake
Examine by gas chromatography (2.2.28), using vigorously for 1 min o Centrifuge if necessary and use the
N,N-diethylaniline R as the internal standard. upper layer.
lnternal standard solution Dissolve 50 mg of The chromatographic procedure may be carried out using:
N,N-diethylamline R in 4 mL of 0.1 M hydrochloric acid and - a glass column 2 m long and 2 mm in internal diameter
dilute to 50 mL with water R. Dilute 1 mL of this solution packed with silanised diatomaceous earth for gas
to 100 mL with water R. chromatography R impregnated with 3 per cent mhn of
Test solution Dissolve in a ground-glass-stoppered tube polymethylphenylsiloxane R,
0.50 g of the substance to be examined in 30.0 mL of - nitrogen f01" chromatography R as the carrier gas at a fiow
water R. Add 1.0 mL of the internal standard solution. rate of 30 mUmin,
Adjust the solution to a ternperature of 26-28 oC.
- a fiame-ionisation detector,
Add 1.0 mL of strong sodium hydroxide solution R and mix
maintaining the temperature of the column at 120 oC and
until completely dissolved. Add 2.0 mL of trimethylpentane R.
that of the injection port and of the detector at 150 oc.
Shake for 2 min and allow the phases to separate. Use the
upper layer. Inject 1 ¡.¡L of the test solution and 1 ¡.¡L of the reference
solution.
Reference solution Dissolve 50.0 mg of
N,N-dimethylaniline R in 4.0 mL of 0.1 M hydrochloric acid
and dilute to 50.0 mL with water R. Dilute 1.0 mL of this
solution to 100.0 mL with water R. Dilute 1.0 mL of this
solution to 30.0 mL with water R. Add 1.0 mL of the O. 2-Ethylhexanoic Acid
internal standard solution and 1.0 mL of strong sodium (Ph . Eur. method 2.4.28)
hydroxide solution R. Add 2.0 mL of trimethylpentane R. Shake Examine by gas chromatography (2.2.28), using
for 2 min and allow the phases to separate. Use the upper 3-cyclohexylpropionic acid Ras the internal standard.
layer. lnternal standard solution Dissolve 100 mg of
The chromatographic procedure may be carried out using: 3-cyclohexylpropionic acid R in cyclohexane R and dilute
- a fused-silica capillary column 25 m long and 0.32 mm in to 100 mL with the same solvento
internal diameter coated with cross-linked Test solution To 0.300 g of the substance to be
polymethylphenylsiloxane R (film thickness 0.52 ¡.¡m), examined, add 4.0 mL of a 33 per cent VIV solution of
- helium for chromatography R as the carrier gas with a split hydrochloric acid R . Shake vigorously for 1 min with 1.0 mL
ratio 1:20, a column head pressure of 50 kPa and a split of the internal standard solution. Allow the phases to
vent of 20 mUmin; separate (if necessary, centrifuge for a better separation).
- a fiame-ionisation detector, Use the upper layer.
- a split-liner consisting of a column about 1 cm long Reference solution Dissolve 75.0 mg of 2-ethylhexanoic
packed with diatomaceous earth for gas chromatography R acid R in the internal standard solution and dilute to
impregnated with 10 per cent m/m of 50.0 mL with the same solution. To 1.0 mL of the solution
poly(dimethyl)siloxane R, add 4.0 mL of a 33 per cent VIV solution of hydrochloric
acid R. Shake vigorously for 1 mino Allow the phases to
maintaining the temperature of the column at 150 oC for
separate (if necessary, centrifuge for a better separation).
5 min, then raising the temperature at arate of 20 oC per
U se the upper layer. '
min to 275 oC and maintaining it at 275 oC for 3 min and
maintaining the temperature of the detector at 300 oC and The chromatographic procedure may be carried out using:
that of the injection port at 220 oc. - a wide-bore fused-silica column 10 m long and 0.53 mm
The retention times are: N,N-dimethylaniline about 3.6 min, in internal diameter coated with macrogol 20 000
N,N-diethylaniline about 5.0 mino 2-nitroterephthalate R (film thickness 1.0 ¡.¡m),
Inject 1 ¡.¡L of the test solution and 1 ¡.¡L of the reference - helium for chromatography R as the carrier gas at a fiow rate
solution. of 10 mUmin,
- a fiame-ionisation detector,
Method B with the following temperature programme:
Examined by gas chromatography (2.2.28), using
naphthalene R as the internal standard.
Time Temperature Rate Comment
lnternal standard solution Dissolve 50 mg of (min) (oC) (OC/min)
naphthalene R in cyclohexane R and dilute to 50 mL with the Column 0·2 40 isothermal
same solvento Dilute 5 mL of this solution to 100 mL with
2·7.3 40 ~ 200 30 linear gradient
cyclohexane R.
Test solution To 1.00 g of the substance to be examined 7.3·10.3 200 isothermal
in a ground-glass-stoppered tube add 5 mL of 1 M sodium Injection port 200
hydroxide and 1.0 mL of the internal standard solution. Detector 300
Stopper the tube and shake vigorously for 1 mino Centrifuge
if necessary and use the upper layer.
Reference solution To 50.0 mg of N,N-dimethylaniline R Inject 1 ¡.¡L of the test solution and 1 ¡.¡L of the reference
add 2 mL of hyd1"ochloric acid R and 20 mL of water R, shake solution.
2014 Appendix VIII P V-A283
The test is not valid unless the resolution between the peaks test sample. If the proteins in solution exist as particles
corresponding to 2-ethylhexanoic acid (first peak) and the comparable in size to the wavelength of the measuring light
internal standard is at least 2.0 . (250 nm to 300 nm), scattering of the light beam results in
Calculate the percentage content of 2-ethylhexanoic acid an apparent increase in absorbance of the test sample.
from the expression: To calculate the absorbance at 280 nm due to light
scattering, determine the absorbances of the test solution at
AT X IR x mR x 2 wavelengths of 320 nm, 325 nm, 330 nm, 335 nm, 340 nm,
AR X IT x mT 345 nm and 350 nm. Plot the logarithm of the observed
absorbance against the logarithm of the wavelength and
AT area of the peak corresponding to 2-ethylhexanoic determine the standard curve best fitting the plotted points
acid in the chromatogram obtained with the test by linear regression. Extrapolate the curve to determine the
solution, logarithm of the absorbance at 280 nm. The antilogarithm of
AR area of the peak corresponding to 2-ethylhexanoic this value is the absorbance attributed to light scattering.
acid in the chromatogram obtained with the Correct the observed values by subtracting the absorbance
reference solution, attributed to light scattering from the total absorbance at
h area of the peak corresponding to the internal 280 nm to obtain the absorbance value of the protein in
standard in the chromatogram obtained with the test solution. Filtration with a 0.2 ¡lm filter that does not adsorb
solution, protein or clarification by centrifugation may be performed
IR area of the peak corresponding to the internal to reduce the effect of light scattering, especially if the
standard in the chromatogram obtained with the solution is noticeably turbid.
reference solution, Calculations Use corrected values for the calculations.
mT mass of the substance to be examined in the test Calculate the concentration of protein in the test solution
solution, in grams, (Cu ) from the following equation:
mR mas s of 2-ethylhexanoic acid in the reference
solution, in grams . Cu = Cs (Au/As)
where Cs is the concentration of protein in the reference
solution and Au and As are the corrected absorbances of the
P. Total Protein test solution and the reference solution, respectively.
(Ph. Eur. method 2.5.33) Method 2

Many of the assay methods described in this chapter can be This method (commonly referred to as the Lowry assay) is
performed using kits from commercial sources. based on the reduction by protein of the
phosphomolybdotungstic mixed acid chromogen in the
Method 1
phosphomolybdotungstic reagent, which results in an
Protein in solution absorbs ultraviolet light at a wavelength of absorbance maximum at 750 nm.
280 nm, due to the presence of aromatic amino acids, mainly The phosphomolybdotungstic reagent reacts primarily with
tyrosine and tryptophan, in the protein structure. This tyrosine residues in the protein. Colour development reaches
property can be used for assay purposes. If the buffer used to a maximum in 20 min to 30 min at room temperature, after
dissolve the protein has a high absorbance relative to that of which there is a gradualloss of colour. Because the method
water, an interfering substance is presentoThis interference is sensitive to interfering substances, a procedure for
may be obviated by using the buffer as compensation liquid precipitation of the protein from the test sample may be
but if the interfering substance produces a high absorbance, used. Most interfering substances cause a lower colour yield;
the results may nevertheless be compromised. At low h~wever, some detergents cause a sliglú increase in colour.
concentrations, protein adsorbed onto the cell may A high salt concentration may cause a precipitate to formo
significantly reduce the content in solution. This can be Because different protein species may give different colour
prevented by preparing samples at higher concentration or by response intensities, the reference substance and test protein
using a non-ionic detergent in the preparation. must be the same. Where separation of interfering substances
Test solution Dissolve a suitable quantity of the substance from the protein in the test sample is necessary, proceed as
to be examined in the prescribed buffer to obtain a solution directed below for interfering substances prior to preparation
having a protein concentration between 0.2 mg/mL and of the test solution. The effect of interfering substances may
2 mg/mL. be minimised by dilution, provided the concentration of the
Reference solution Prepare a solution of a suitable test protein remains sufficient for accurate measurement.
reference substance for the protein to be determined, in the Use distilled water R to prepare all buffers and reagents used
same buffer and at the same protein concentration as the test for this method.
solution. Test solution Dissolve a suitable quantity of the substance
Procedure Keep the test solution, the reference solution to be examined in the prescribed buffer to obtain a solution
and the compensatiori liquid at the same temperature during having a concentration within the range of the standard
the performance of this test. Determine the absorbances curve. A suitable buffer will produce a solution of
(2.2.25) of the test solution and the reference solution in pH 10.0 to 10.5.
quartz cells at 280 nm, using the prescribed buffer as the Reference solutions Dissolve the reference substance for
compensation liquid. The response must be linear in the the protein to be determined in the prescribed buffer. Dilute
range of protein concentrations to be assayed to obtain portions of this solution with the same buffer to obtain not
accurate results. fewer than five reference solutions having protein
Light scattering The accuracy of the determination of concentrations evenly spaced over a suitable range situated
protein can be diminished by the scattering of light by the between 5 ¡lg/mL and 100 ¡lg/mL.
V -A284 Appendix VIII P 2014
Blank Use the buffer used to prepare the test solution and Reference solutions Dissolve the reference substance for
the reference solutions. the protein to be determined in the prescribed buffer. Dilute
Copper sulfate reagent Dissolve 100 mg of copper portions of this solution with the same buffer to obtain not
sulfate R and 0.2 g of sodium tartrate R in distilled water R and fewer than five reference solutions having protein
dilute to 50 mL with the same solvent. Dissolve 10 g of concentrations evenly spaced over a suitable range situated
anhydrous sodium carbonate R in distilled water R and dilute to between 0.1 mg/mL and 1 mg/mL.
50 mL with the same solvent. Slowly pour the sodium Blank Use the buffer used to prepare the test solution and
carbonate solution into the copper sulfate solution with the reference solutions.
mixing. Use within 24 h. Acid blue 90 reagent Dissolve 0.10 g of acid blue 90 R in
Alkaline copper reagent Mix 1 volume of copper sulfate 50 mL of alcohol R. Add 100 mL of phosphoric acid R, dilute
reagent, 2 volumes of a 50 gIL solution of sodium dodecyl to 1000 mL with distilled water R and mix. Filter the solution
sulfate R and 1 volume of a 32 gIL solution of sodium and store in an amber bottle at room temperature. Slow
hydroxide R . Store at room temperature and use within precipitation of the dye occurs during storage. Filter the
2 weeks. reagent before using.
Diluted phosphomolybdotungstic reagent Mix 5 mL of Procedure Add 5 mL of acid blue 90 reagent to
phosphomolybdotungstic reagent R with 55 mL of distilled 0.100 mL of each reference solution, of the test solution and
water R. Store in an amber bottle, at room temperature. of the blank. Mix by inversion. Avoid foarning, which will
Procedure To 1.0 mL of each reference solution, of the lead to poor reproducibility. Determine the absorbances
test solution and of the blank, add 1.0 mL of alkaline copper (2.2.25) of the standard solutions and of the test solution at
reagent and mix. Allow to stand for la mino Add 0.5 mL of 595 nm, using the blank as compensation liquido Do not use
the diluted phosphomolybdotungstic reagent, mix and allow quartz (silica) spectrophotometer cells because the dye binds
to stand at room temperature for 30 min. Determine the to this material.
absorbances (2.2.25) ofthe solutions at 750 nm, using the Calculations The relationship of absorbance to protein
solution from the blank as compensation liquido concentration is non-linear; however, if the range of
Calculations The relationship of absorbance to protein concentrations used 10 prepare the standard curve is
concentration is non-linear; however, if the range of sufficiently small, the latter will approach linearity. Plot the
concentrations used to prepare the standard curve is absorbances of the reference solutions against protein
sufficiently small, the latter will approach linearity. Plot the concentrations and use linear regression 10 establish the
absorbances of the reference solutions against the protein standard curve. From the standard curve and the absorbance
concentrations and use linear regression to establish the of the test solution, determine the concentration of protein in
standard curve. From the standard curve and the absorbance the test solution.
of the test solution, determine the concentration of protein in
the test solution. Method 4
Interfering substances In the following procedure, This method (commonly referred 10 as the bicinchoninic acid
deoxycholate-trichloroacetic acid is added to a test sample to or BCA assay) is based on reduction of the cupric (Cu 2 +) ion
remove interfering substances by precipitation of proteins to cuprous (Cu 1+) ion by protein. The bicinchoninic acid
before determination; tilis technique can also be used to reagent is used to detect the cuprous ion. Few substances
concentrate proteins from a dilute solution. interfere with the reaction. When interfering substances are
present their effect may be minimised by dilution, provided
Add 0.1 mL of a 1.5 gIL solution of sodium deoxycholate R to
that the concentration of the protein to be,determined
1 mL of a solution of the substance to be examined.
remains sufficient for accurate measurement. Alternatively,
Mix using a vortex mixer and allow to stand at rooP1
the protein precipitation procedure given in Method 2 may
temperature for la min. Add 0.1 mL of a 720 gIL solution
be used to remove interfering substances. Because different
of trichloroacetic acid R and mix using a vortex mixer.
protein species may give different colour response intensities,
Centrifuge at 3000 g for 30 min, decant the liquid and
the reference protein and protein to be determined must be
remove any residual liquid with a pipette. Redissolve the
tbe same.
protein pellet in 1 mL of alkaline copper reagent.
Use distilled water R to prepare all buffers and reagents used
Method 3 for this method.
This method (commonly referred to as the Bradford assay) is Test solution Dissolve a suitable quantity of the substance
based on the absorption shift from 470 nm to 595 nm to be examined in the prescribed buffer 10 obtain a solution
observed when the acid blue 90 dye binds to protein. having a concentration within the range of the concentrations
The acid blue 90 dye binds most readily to arginine and of the reference solutions.
Iysine residues in the protein which can lead to variation in Reference solutions Dissolve the reference substance for
the response of the assay to different proteins. The protein thé pro te in 10 be determined in the prescribed buffer. Dilute
used as reference substance must therefore be the same as portions of this solution with the same buffer to obtain not
the protein to be determined. There are relatively few fewer than five reference solutions having protein
interfering substances, but it is preferable to avoid detergents concentrations evenly spaced over a suitable range situated
and ampholytes in the test sample. Highly alkaline samples between 10 ¡¡g/mL and 1200 ¡¡g/mL.
may interfere with the acidic reagent. Blank Use the buffer used to prepare the test solution and
Use distilled water R to prepare all buffers and reagents used the reference solutions .
for this method. BCA reagent Dissolve lag of disodiw11 bici11choninate R,
Test solution Dissolve a suitable quantity of the substance 20 g of sodium carbonate m0110hydrate R, 1.6 g of sodium
to be examined in the prescribed buffer to obtain a solution tartrate R, 4 g of sodium hydroxide R, and 9.5 g of sodium
having a concentration within the range of the standard hydrogen carbonate R in distilled water R. Adjust, if necessary,
curve. to pH 11.25 with a solution of sodium hydroxide R or a
2014 Appendix VIII P V-A285
solution of sodium hydrogen carbonate R. Dilute to 1000 mL stand at a temperature between 15 oC and 25 oC for not less
with distilled water R and mix. than 15 mino Within 90 min of addition of the biuret
Copper-BCA reagent Mix 1 mL of a 40 giL solution of reagent, determine the absorbances (2.2.25) of the reference
copper sulfate R and 50 mL of BCA reagent. solutions and of the test solution at the maximum at
Procedure Mix 0.1 mL of each reference solution, of the 545 nm, using the blank as compensation liquid.
test solution and of the blank with 2 mL of the copper-BCA Any solution that develops turbidity or a precipita te is not
reagent. Incubate the solutions at 37 oC for 30 min, note the acceptable for calculation of protein concentration.
time and allow the mixtures to cool to room temperature. Calculations The relationship of absorbance to protein
Within 60 min of the end of incubation, determine the concentration is approximately linear within the indicated
absorbances (2.2.25) of the reference solutions and of the range of protein concentrations for the reference solutions.
test solution in quartz cells at 562 nm, using the blank as Plot the absorbances of the reference solutions against
compensation liquido After the solutions have cooled to room protein concentrations and use linear regression to establish
temperature, the colour intensiry continues to increase the standard curve. Calculate the correlation coefficient for
gradually. the standard curve. A suitable system is one that yields a line
Calculations The relationship of absorbance to protein having a correlation coefficient not less than 0.99. From the
concentration is non-linear; however, if the range of standard curve and the absorbance of the test solution,
concentrations used to prepare the standard curve is determine the concentration of protein in the test solution.
sufficiently small, the latter will approach lineariry. Plot the Interfering substances To minimise the effect of
absorbances of the reference solutions against protein interfering substances, the protein can be precipitated from
concentrations and use linear regression to establish the the test sample as follows: add 0.1 volumes of a 500 giL
standard curve. From the standard curve and the absorbance solution of trichloroacetic acid R to 1 volume of a solution of
of the test solution, determine the concentration of protein in the test sample, withdraw the supernatant layer and dissolve
the test solution. the precipitate in a small volume of 0.5 M sodium hydroxide.
Use the solution obtained to prepare the test solution.
Method 5
This method (cornrnonly referred to as the biuret assay) is
Method 6
based on the interaction of cupric (Cu 2 +) ion with protein in This fiuorimetric method is based on the derivatisation of the
alkaline solution and resultant development of absorbance at protein with o-phthalaldehyde, which reacts with the primary
545 nm. This test shows minimal difference between amines of the protein (N-terminal amino acid and the
equivalent IgG and albumin samples. Addition of the sodium 8-amino group of Iysine residues) . The sensitivity of the assay
hydroxide and the biuret reagent as a combined reagent, can be increased by hydrolysing the protein before adding
insufficient mixing after the addition of the sodium o-phthalaldehyde . Hydrolysis makes the o:-amino group of
hydroxide, or an extended time between the addition of the the constituent amino acids available for reaction with the
sodium hydroxide solution and the addition of the biuret phthalaldehyde reagent. The method requires very small
reagent will give IgG samples a higher response than albumin quantities of the protein. Primary amines, such as
samples. The trichloroacetic acid method used to minimise tris(hydroxyrnethyl)aminomethane and amino acid buffers,
the effects of interfering substances also can be used to react with phthalaldehyde and must be avoided or removed.
determine the protein content in test samples at - Arnmonia at high concentrations reacts with phthalaldehyde.
concentrations below 500 ¡.¡glmL. The fiuorescence obtained when amine reacts with
U se distilled water R to prepare all buffers and reagents used phthalaldehyde can be unstable. The use of automated
for this method. procedures to standardise this procedure may improve the
accuracy and precision of the test.
Test solution Dissolve a suitable quantiry of the substance
to be examined in a 9 gIL solution of sodium chloride R to Use distilled water R to prepare all buffers and reagents used
obtain a solution having a concentration within the range of for this method.
the concentrations of the reference solutions. Test solution Dissolve a suitable quantity of the substance
Reference solutions Dissolve the reference substance for to be examined in a 9 gIL solution of sodium chloride R to
the protein to be determined in a 9 giL s~lution of sodium obtain 'a solution having a concentration within the range of
chloride R . Dilute portions of this solution with a 9 gIL the concentrations of the reference solutions. Adjust the
solution of sodium chloride R to obtain not fewer than three solution to pH 8 to 10.5 before addition of the
reference solutions having protein concentrations evenly phthalaldehyde reagent.
spaced over a suitable range situated between 0.5 mglmL Reference solutions Dissolve the reference substance for
and 10 mglmL. the protein to be determined in a 9 giL solution of sodium
Blank Use a 9 giL solution of sodium chloride R. chloride R. Dilute portions of this solution with a 9 gIL
solution of sodium chloride R to obtain not fewer than five
Biuret reagent Dissolve 3.46 g of copper sulfate R in
reference solutions having protein concentrations evenly
10 mL of hot distilled water R, and allow to cool (Solution
spaced over a suitable range situated between 10 ¡.¡glmL and
A). Dissolve 34.6 g of sodium citrate R and 20.0 g of
200 ¡.¡g/mL. Adjust the solutions to pH 8 to 10.5 before
anhydrous sodium carbonate R in 80 mL of hot distilled
addition of the phthalaldehyde reagent.
water R, and allow to cool (Solution B). Mix solutions A
and B and dilute to 200 mL with distz7led water R. Blank solution Use a 9 gIL solution of sodiwn chloride R.
Use within 6 months. Do not use the reagent if it develops Borate buffer solution Dissolve 61.83 g of bon'c acid R in
turbidiry or contains any precipita te. distilled water R and adjust to pH 10.4 with a solution of
Procedure To one volume of the test solution add an potassium hydroxide R. Dilute to 1000 mL with distilled
equal volume of a 60 gIL solution of sodium hydroxide R and water R and mix.
mix. Immediately add biuret reagent equivalent to Phthalaldehyde stock solution Dissolve 1.20 g of
0.4 volumes of the test solution and mix rapidly. Allow to phthalaldehyde R in 1.5 mL of methanol R, add 100 rnL of
V -A286 Appendix VIII Q 2014
borate buffer solution and mix. Add 0.6 mL of a 300 gIL adapted, depending on the expected amount of acetic acid in
solution of macrogol 23 lauryl ether R and mix. Store at room the sample.
temperature and use within 3 weeks. Reference solution Prepare a 0.10 gIL solution of glacial
Phthalaldehyde reagent To 5 mL of phthalaldehyde acetic acid R in a mixture of 5 volumes of mobile phase B
stock solution add 15 f!L of 2-mercaptoethanol R. Prepare at and 95 volumes of mobile phase A.
least 30 min before use. Use within 24 h. The chromatographic procedure may be carried out using:
Procedure Mix 10 f!L of the test solution and of each of - a stainless steel colurnn 0.25 m long and 4.6 mm in
the reference solutions with 0.1 mL of phthalaldehyde internal diameter packed with octadecylsilyl silica gel for
reagent and allow to stand at room temperature for 15 mino chromatography R (5 f!m),
Add 3 mL of 0.5 M sodium hydroxide and mix. Determine - as mobile phase at a flow rate of 1.2 mUmin:
the fluorescent intensities (2.2.21) of solutions from the
reference solutions and from the test solution at an excitation Mobile phase A Dilute 0.7 mL of phosphon'c acid R to
wavelength of 340 nm and an emission wavelength between 1000 mL with water R; adjust the pH to 3.0 with strong
sodium hydroxide solution R,
440 and 455 nm. Measure the fluorescent intensity of a
given sample only once, since irradiation decreases the Mobile phase B methanol R2,
fluorescence intensity.
Calcu1ations The relationship of fluorescence to protein Time Mobile phase A Mobile phase B
concentration is linear. Plot the fluorescent intensities of the (min) (per cent V!JI) (per cent V!JI)
reference solutions against protein concentrations and use 0·5 95 5
linear regression to establish the standard curve. From the 5 ·10 95 --7 50 5 --7 50
standard curve and the fiuorescent intensity of the test 10·20 50 50
solution, determine the concentration of protein in the test
solution. 20·22 50 --7 95 50 --7 5
22·30 95 5
Method 7
This method is based on nitro gen analysis as a means of - as detector a spectrophotometer set at 210 nm.
protein determination. Interference caused by the presence of
other nitrogen-containing substances in the test sample can Inject 10 f!L of the reference solution and 10 ¡!L of the test
affect the determination of protein by this method. Nitrogen solution. In the chromatograms obtained, the peak
analysis techniques destroy the test sample during the corresponding to acetic acid has a retention time of 3-4 mino
analysis but are not limited to protein presentation in an The baseline presents a steep rise after the start of the linear
aqueous environment. gradient, which corresponds to the elution of the peptide
from the colurnn. Determine the content of acetic acid in the
Procedure A Proceed as prescribed for the determination peptide.
of nitrogen by sulfuric acid digestion (2.5.9) or use
commercial instrumentation for Kjeldahl nitrogen assay.
Procedure B Commercial instrurnentation is available for
nitrogen analysis. Most nitrogen analysis instrurnents use
R. Nickel in Hydrogenated Vegetable Oils
pyrolysis (i.e. combustion of the sample in oxygen at (Ph. Eur. method 2.4.31)
temperatures approaching 1000 OC), which produces ni trie Atomic absorption spectrometry (2.2.23, Method J) .
oxide (NO) and other oxides of nitrogen (NO,,) from the CAUTION: when using ctosed high-pressure digestion vessels and
nitrogen present in the substance to be examined. Sorne microwave laboratory ovens, be familiar with the safety and
instruments convert the nitric oxides to nitro gen gas, which operating instructions given by the manufacturero
is quantified using a thermal-conductivity detector. Other The reagents magnesium nitrate R and ammonium dihydrogen
instruments mix nitric oxide (NO) with ozone (0 3) to phosphate R must be controlled for nickel before use. The actual
produce excited nitrogen dioxide (N0 2*), which emits light nickel content is taken into account in the calculation of the nickel
when it decays and can be quantified with a content of the sample.
chemiluminescence detéctor. A protein reference material
Test solution Weigh 0.250 g (m) of the substance to be
that is relatively pure and is similar in composition to the
examined into a suitable high-pressure-resistant digestion
test proteins is used to optimise the injection and pyrolysis
vessel (fiuoropolymer or quartz glass), add 6.0 mL of nickel-
parameters and to evaluate consistency in the analysis.
free nitric acid R and 2.0 mL of strong hydrogen peroxide
Calcu1ations The protein concentration is calculated by solution R Prepare a blank solution in the same manner.
dividing the nitro gen content of the sample by the known Place the c10sed ves seis in a laboratory microwave oven and
nitrogen content of the protein. The known nitrogen content digest with an appropriate programme, e.g. 1000 W for
of the protein can be determined from the chemical 40 mino Allow the digestion vessels to cool before opening.
composition of the protein or by comparison with a suitable Add 2.0 mL of strong hydrogen peroxide solution R and repeat
reference substance the digestion step. Allow the digestion ves seis to cool before
opening. Quantitatively transfer to a 25 mL fiask, add
0.5 mL of a 10 gIL solution of magnesium nitrate R and
0.5 mL of a 100 gIL solution of ammonium dihydrogen
Q. Acetic Acid in Synthetic Peptides phosphate R, dilute to 25.0 mL with water for
(Ph. Eur. method 2.5.34) chromatography R and mix.
Examine by liquid chromatography (2.2.29) . Reference solutions Into 4 volurnetric flasks, introduce
Test solution Prepare as described in the monograph. 25 f!L, 50 f!L, 75 f!L and 100 f!L of nickel standard solution
The concentration of peptide in the solution may be (5 ppm Ni) R . To each fiask, add 0.5 mL of a 10 gIL
solution of inagnesium nitra te R, 0.5 mL of a 100 giL
2014 Appendix VIII S V-A287
solution of ammonium dihydragen phosphate R and 6.0 mL of and isopropyl methanesulfonate R (IMS) in the internal
niekel-free nitrie acid R and dilute to 25 .0 mL with water far standard solution and dilute to 50.0 mL with the same
ehramatography R . Mix to obtain reference solutions solution. Dilute 74 ¡.tL ofthe solution to 10.0 mL with the
containing respectively 5 nglmL, 10 ng/mL, 15 nglrnL and internal standard solution. Dilute 100 ~lL of this solution to
20 nglmL (Ppb) of nickel. 10.0 mL with the internal standard solution.
Zero solution In a volumetric ftask, introduce 1.0 mL of a Referenee solution (b)Dilute 3.0 mL of reference solution
10 giL solution of magnesium nitrate R, 1.0 mL of a 100 gIL (a) to 10.0 mL with the internal standard solution.
solution of ammonium dihydragen phosphate R and 12.0 mL of Column:
niekel-free nitrie acid R. Dilute to 50.0 mL with water far
- matenal: fused silica;
ehromatography R and mix.
- size: 1 = 15 m, el = 0.25 mm;
Method - stationary phase: poly(dimethyl)siloxane R (film thickness
Determine the absorbance of each solution at 232.0 nm 1 ¡.tm).
using a suitable graphite furnace atomic absorption Carrier gas helium for ehromatography R .
spectrometer equipped with a background compensation Flow rate 1 mUmin.
system, a pyrolytically-coated tube, and a nickel hollow- Pulsed splitless 250 kPa, 0.25 mino
cathode lampo Maintain the drying temperature of the
Temperature:
fuma ce at 120 oC for 35 s after a 5 s ramp, the ashing
temperature at 1100 oC for 10 s after a 30 s ramp, the
cooling temperature at 800 oC for 5 s after a 5 s decrease, Time Temperature
(min) (OC)
and the atomisation temperature at 2600 oC for 7 S. Use the
zero solution to set the instrument to zero. Using the Column 0-1 55
calibration curve, determine the concentrations of the test 1-9 55 -) 135
solution and the blank solution from the corresponding
1njection port 240
absorptions. If necessary, dilute with the zero solution to
obtain a reading within the calibrated absorbance range. Detector : transfer line 280
Calculate the content of Ni in micrograms per gram (ppm) so urce 230
using the following expression:
analyser 150
e X f
m x 40 Detectian mass spectrometer as described below; adjust the
detector settings so as to comply with the system suitability
e measured concentration of Ni, in nanograms per criteria:
millilitre; - quadrupole mass spectrometer equipped with an electron
f dilution factor of the rest solution; impact ionisation mode (70 eV);
m mass of the substance to be examip.ed, in grams.
- mas s spectrometer parameters for the fragmentometric
mode (single-ion monitoring (SIM)) set as follows:
S. Methyl, Ethyl and Isopropyl Substance miz Duration of monitoring
Methanesulfonate in Methanesulfonic Butyl methanesulfonate (BMS) 56

IR between 7.0 min
and 9.0 min
Acid Methyl methanesulfonate (MMS) 80
IR between 2.0 min
and 3.5 min
(Ph. Eur. method 2.5.37) IR between 4.0 min
Ethyl methanesulfonate (EMS) 79 and 4.7 min
The following method has been validated for the tnethyl,
ethyl and isopropyl esters of methanesulfonic acid at Isopropyl methanesulfonate (IMS) 123
IR between 4.7 min
concentrations in the range of 0.5 ppm to 100 ppm . and 5.5 min
If it is intended to be used to determine levels of

methanesulfonic acid esters outside this validated range, for Injeetion 2 ¡.tL.
example in early steps of the synthesis prior to their removal, Relative retention With reference to the internal standard
the concentration of the test solution has to be adjusted (BMS) (retention time = about 7.6 min) :
accordingly. MMS = about 0.3; EMS = about 0.5; IMS = about 0.6.
Gas chromatography (2.2.28) coupled with mass System suitability:
spectrometry (2.2.43) . - resolution: minimum 3.0 between the peaks due to EMS
Internal standard solutian Dilute 7 ¡.tL of bueyl and IMS in the chromatogram obtained with reference
methanesulfonate CRS (BMS) to 10.0 mL with methylene solution (a);
ehloride R. Dilute 10 ¡.tL of the solution to 100.0 mL with - signal-to-noise ratio: minimum 10 for the peaks due to
methylene ehloride R. MMS, EMS and IMS in the chromatogram obtained with
Test solutian Add 0.74 g of the substance to be examined to reference solution (b).
10.0 mL ofwater R and extract with 10.0 mL ofthe internal Calculate the content of MMS, EMS or IMS in pans per
standard solution. Allow to separare and transfer the organic million using the following expression:
layer to a vial containing anhydrous sodium sulfate R. Shake
and filter. A2 X h X Wl X e x 0.148
Referenee solution (a) Dissolve 50 mg each of methyl Al x h X W2
methanesulfonate R (MMS), ethyl methanesulfonate R (EMS)
V-A288 Appendix VIII T 2014
area of the peak due to MMS, EMS or IMS in Referenee solution (e) Dilute 500 J..lL of reference
the chromatogram obtained with reference solution (a) to 20.0 mL with the internal standard solution.
solution (a); Introduce 0.50 mL of this solution and 0.50 mL of solution
area of the peak due ro MMS, EMS or IMS in A into a 20 mL headspace vial and seal the vial immediately
the chromarogram obtained with the test solution; with a polytetrafluoroethylene-coated silicon membrane and
percentage content of MMS, EMS or IMS; an aluminium cap.
area of the peak due ro the internal standard in Column:
the chromatogram obtained with reference - material: fused silica;
solution (a);
- size: 1 = 30 m, 0 = 0.25 mm;
area of the peak due to the internal standard in
- stationary phase: polar-deactivated polyethyleneglycol R (film
the chromatogram obtained with the test solution;
thickness 1 J..lm).
mass of MMS, EMS or IMS used ro prepare
reference solution (a), in milJigrams; Carrier gas helium for chromatography R.
mass of the substance to be examined in the test Flow rate 0.5 mUmin.
solution, in milligrams; Split ratio 1:20.
0.148 dilution factor.
- equilibration time: 30 min;
T. Methyl, Ethyl and Isopropyl - lransfer-line temperalure: 120 oc.
Methanesulfonate in Active Substances Temperature:

Time Temperature
The folJowing general method has been validated for the (oC)
(min)
determination of methyl, ethyl and isopropyl esters of
Column 0·1 40
methanesulfonic acid (in concentrations between 0.2 ppm
and 5 ppm) in betahistine mesilate. l· 10 40 ~ 130
If it is intended ro use the method for other active Injection port 220
substances, particularly those that contain different Detector transfer line 280
concentrations of the methanesulfonic acid esters, the
source 250
concentrations of the test solution and reference solution
must be adjusted accordingly and the method must be analyser 200
suitably validated.
Method At the end of analysis the temperature of the column is

raised to 240 oC and maintained at this temperature for
Head-space gas chromatography (2.2.28) coupled with mass
spectrometry (2.2.43). Prepare the test solution and reference 7 mino
solutions immediately before use. Deteetion Mass spectrometer as described below; adjust
the detector settings so as to comply with the system
Solvent mixture water R, acetonitrile R (20:80 V/V).
suitability criteria; alternatively a suitable electron-capture
Solution A Dissolve 30 mg of anhydrous sodium detector may be used:
thiosulfate R and 60.0 g of sodium iodide R in water R and
- quadrupole mass spectrometer equipped with an electron
dilute to 50.0 mL with the same solvent.
impact ionisation mode (70 eV);
Internal standard solution Dilute 10 J..lL of butyl
methanesulfonate CRS (BMS) ro 10.0 mL with the solvent
- mass spectrometer parameters for the fragmentometric
mode (single-ion monitoring (SIM)) set as folJows:
mixture. Dilute 20 J..lL of the solution to 10.0 mL with the
solvent mixture. DlIute 10.0 mL of this solution ro
100.0 mL with the solvent mixture. Quantitation ion Qualification ion
Substance
Test solution Weigh 25 .0 mg of the substance to be (miz) (miz)
examined inro a 20 mL headspace vial, add 0.50 mL of Butyl iodide (Bul)O 184 127
solution A and 0.50 mL of the internal standard solution, Methyl iodide (Mel)° 142 127
then seal the vial immediately with a polytetrafiuoroethylene-
coated siJicon Ínembrane and an aluminium cap. Following Ethyl iodide (EtI)O 156 127
the derivatisation reaction, a precipitate may be observed, however Isopropyl iodide (iPrI)O 170 127
this does not affect the validity of the quantification.
° formed from BMS, MMS, EMS and IMS in the derivatisation reaction.
Referenee solution (a) Dissolve 25.0 mg each of methyl
methanesulfonate R (MMS), ethyl methanesulfonate R (EMS)
and isopropyl methanesulfonate R (IMS) in toluene R and diJute
Injeetion 1 mL of the gas phase of the test solution and
reference solutions (b) and (c).
ro 5.0 mL with the same solvent. Dilute 50 J..lL of the
solution ro 25.0 mL with the internal standard solution. Relative retention With reference to the internal standard
(BuI) (retention time = about 8.5 min): MeI = about 0.51;
Referenee solution (b) Dilute 20 J..lL of reference solution
EtI = about 0.63; iPrI = about 0.68.
(a) ro 20.0 mL with the intemal standard solution. Introduce
0.50 mL of this solution and 0.50 mL of solution A into a System suitability:
20 mL headspace vial and se al the vial immediately with a - resolution: minimum 1.5 between the peaks due to EtI and
polytetrafiuoroethylene-coated siJicon membrane and an iPrI in the chromatogram obtained with reference solution
aluminium cap. (c);
2014 Appendix VIII V V-A289
- signal-to-noise ratio: minimum 10 for the peak due 10 each Carrier gas helium for ehromatography R.
alkyl iodide in the chromatogram obtained with reference Flow rate 1 mLJmin.
solution (b).
Pulsed splitless 60 kPa, 0.1 mino
Calculate the content in parts per million of each alkyl
Temperature:
methanesulfonate using the following expression:
A2 X h X Wl X e x 0.05 Time Temperature

(mio) (OC)
Al x h X W2
Column 0-4 40
are a of the peak due 10 each alkyl iodide in the 4-8 40 --t 200
chromatogram obtained with reference solution (c); Injection port 240

area of the peak due 10 each alkyl iodide in the Detector: transfer line 280
chromatogram obtained with the test solution;
source 230
C percentage content of each ester;
JI area of the peak due to the internal standard in the analyser 150
chromatogram obtained with reference solution (c);
area of the peak due 10 the internal standard in the
chromatogram obtained with the test solution; At the end of analysis the temperature of the eolumn is
mass of each ester used 10 prepare reference raised 10 270 oC and maintained at this temperature for
solution (a), in milligrams; 8 mino
mass of the substance to be examined in the test Detection Mass spectrometer as described below; adjust
solution, in milligrams; the detector settings so as to eomply with the system
0.05 dilution factor. suitability eriteria:
- quadrupole mass spectrometer equipped with an electron
impact ionisation mode (70 eV);
- mass spectrometer parameters for the fragmentometric
V. Methanesulfonyl Chloride in mode (single-ion monitoring (SIM)) set as follows :
Methanesulfonic Acid
(Ph. Eur. method 2.5.39) Substaoce miz Duration of monitoring
The following method has been validated for the Methanesulfonyl
79 IR between 3.3 min and 6.0 min
determination of methanesulfonyl chloride in chloride
methanesulfonic acid at concentrations in the range of Butyl methane-
56 IR between 6.0 min and S.O min
0.05 ppm to 50 ppm. sulfonate (BMS)
Gas chromatography (2.2.28) couplea with mas s

spectrometry (2.2.43) . Injection 5 ¡.tL of the test solution, reference solutions (b)
Internal standard solution Dissolve 7 ).lL of butyl and (e), the internal standard solution and methylene
ehloride R .
methanesulfonate CRS (BMS) in methylene ehloride R and
dilute 10 10.0 mL with the same solvent. Dilute 5.0 mL of Relative retention With reference to the internal standard
this solution to 50.0 mL with methylene ehloride R. (BMS) (retention time = about 7.2 min):
Test solution To 5 mL of water R, add 7.4 g of the methanesulfonyl chloride = about 0.68.
substance to be examined and mix slowly. After cooling, add System suitability:
5.O mL of methylene ehloride R ¡¡nd 100 ~LL of the internal - in the chromatogram obtained with the internal standard
standard solution and shake. Allow to separate and transfer solution, there is no peak with the same retention time as
the organic layer 10 a vial containing 1 g of anhydrous sodium methanesulfonyl ehloride;
sulfate R . Repeat the extraction twice with 5.0 mL of - resolulÍon: minimum 5.0 between the peaks due to
methylene ehloride R each time, combine the organic layers methanesulfonyl chloride and BMS in the ehromatogram
and filter. obtained with reference solution (b);
Reference solution (a) Dissolve 50.0 mg of - signal-to-noise ratio: minimum 10 for the peak due 10
methanesulfonyl ehloride R in methylene ehloride R and dilute to methanesulfonyl ehloride in the chromatogram obtained
10.0 mL with the same solvent. Dilute 1.0 mL of the with reference solution (c) .
solution to 10.0 mL with methylene ehloride R. Dilute 300 ¡.tL Calculate the content of methanesulfonyl chloride in parts
of this solution 10 10.0 mL with methylene ehloride R . per million using the following expression:
Reference solution (b) Dilute 500 ¡.tL of reference
solution (a) and 100 ).lL of the internal standard solution to A2 X h X Wl X e x 1.5
15. O mL with methylene ehloride R.
Al x [2 X W2
Referenee solution (e) Dilute 25 ¡.LL of reference solution
(a) and 100 ¡.LL ofthe internal standard solution to 15.0 mL
with methylene ehloride R. area of the peak corresponding to methanesulfonyl
chloride in the chromatogram obtained with
Column:
referenee solution (b);
- material: fused silica; area of the peak corresponding to methanesulfonyl
- size: 1 = 15 m, 0 = 0.25 mm; chloride in the chromatogram obtained with the test
- stationary phase: poly(dimethy1)siloxane R (film thickness solution;
1 ¡.Lm). C percentage eontent of methanesulfonyl chloride;
V-A290 Appendix VIII W 2014
11 area of the peak corresponding to BMS in the preparation andlor measurement method must be developed
chromatogram obtained with reference solution (b); and validated (se e Figures 2.4.20.-1 and 2.4.20.-2).
area of the peak corresponding to BMS in the Sample preparation
chromatogram obtained with the test solution;
Sample preparation is critical to the success of elemental
mass of methanesulfonyl chloride used to prepare
analysis. Many techniques not using direct measurement are
reference solution (a), in milligrams;
heavily dependent on sample transport.
mass of the sample in the test solution, in milligrams;
dilution factor. If an atomisation system is used, the most conventional
means by which samples are introduced into the atomisation
system is by solution nebulisation. In this case, solid samples
must be dissolved in order to be introduced into the
W. Determination of Metal Catalyst or atomisation system. Samples may be dissolved in any
appropriate solvent. The use of aqueous or dilute nitric acid
Metal Reagent Residues 1 solutions is strongly recommended, due to minimal
(Ph. Eur. method 2.4.20) interference with these solvents compared to other solvents.
Hydrochloric acid, hydroftuoric acid, perchloric acid, sulfuric
Introduction acid and hydrogen peroxide, at various concentrations, can
This chapter describes the general approach for the be used to dissolve the samples. The viscosity of sulfuric acid
determination of metal catalyst or metal reagent residues in is greater than that of the other acids and is to be taken into
substances for pharmaceutical use. As the chemical account as it can affect the overall ftuidity of the solution.
composition of the considered substances and the The choice of solvents inc1udes, but is not limited to, the use
specification limits for the metal(s) of interest vary of dilute bases, straight or diluted organic solvents,
considerably, it is not possible to describe all suitable sample combinations of acids or bases, and combinations of organic
preparation and measurement methods. Therefore, any solvents. Acids, bases, and hydrogen peroxide of high purity
method that fulfils the requirements described in this chapter must be used, especially when ICP-MS is employed.
may be used. For aqueous solutions, use deionised distilled water R. Diluents
The results of the analysis are acceptable only if the system must be checked for interference if they are used in an
suitability has been demonstrated by a suitable test. Before analysis. Because it is not always possible to obtain organic
the initial use of a method, the analyst must ensure that the solvents that are free of metal s, organic solvents of the
method is appropriate for the samples and instruments used. highest purity possible with regard to metal contaminants
This is accomplished by applying a validation procedure to must be used. Specifically for ICP techniques, where samples
methods not described in the specific monograph or by a are introduced into the plasma via solution nebulisation, it is
system suitability test for methods described in the important to consider the potential matrix effects and
monograph. Decision trees for the choice of the sample interferences that might arise from the solvent. The use of an
preparation and the measurement procedures are presented appropriate internal standard andlor matching the standard
in Figures 2.4 .20.-1 and 2.4.20.-2. matrix with samples should be applied for ICP-AES and
ICP-MS analyses in cases where accuracy and precision are
Procedures not sufficient. In any case, the selection of an appropriate
As a reference procedure is not provided for each metal, internal standard should take into account the metal(s) of
matrix and concentration, the choice of procedure according interest, ionisation energy, wavelengths or masses, and the
to Figures 2.4.20.-1 and 2.4.20.-2, inc1uding sample nature of the sample matrix.
preparation, detection technique and instrument parameters, Where a sample is found not to be soluble in any acceptable
is the responsibility of the user. solvent, a variety of digestion or incineration techniques can
Use the ftow chartin Figure 2.4.20.-1 to define the sample be employed. These inc1ude hot-plate digestion, incineration
preparation method and the ftow chart in Figure 2.4.20.-2 to and microwave-assisted digestions, using open- and c1osed-
define the measurement method. The sample preparation vessel.
method should yield a sufficient quantity of sample to allow The decision regarding the type of digestion technique to be
quantification of each metal at the specified limit stated in used depends on the nature of the sample being digested, as
the specific monograph or the general chapter. weH as on the metal(s) of interest and the concentration
AH suitable sample preparation methods and measurement range of the metals to be quantified. Open-vessel digestion is
techniques (e.g. 2.2.22. Atomic emission spectrometry (AES), not recommended for the analysis of volatile metals.
2.2.23. Atomic absorption spectrometry (AAS), 2.2.37. X-ray The suitability of a digestion technique, whether open- or
fiuorescence spectrometry (XRFS), 2.2.57. 1nductively coupled c1osed-vessel, should be supported by spike recovery
plasma-atomic emission spectrometry (ICP-AES), 2.2.58. experiments in order to verify that, within an acceptable
1nductively coupled plasma-mass spectrometry (ICP-MS), 2.4.2. tolerance, volatile metals have not been lost during sample
Arsenic, 2.4.8. Heavy metals, 2.4.9. 1ron, 2.4.10. Lead in preparation. The digestion cyc1e is suitable if a c1ear solution
sugars, 2.4.15. Nickel in polyols, 2.4.31. Nickel in hydrogenated is obtained.
vegetable oils) can be used for the determination of metal It is important to consider the selection of the type, the
residues, if the method has been verified before the initial use material of construction, the pretreatrnent, and the c1eaning
by a system suitability test or a validation procedure of analytical labware used in elemental analyses. The material
according to this chapter. must be inert and, depending on the specific application,
If no sample preparation and/or measurement method is resistant to caustics, acids, andlor organic solvents. For sorne
described in the specific monograph, a suitable sample analyses, care must be exercised to prevent the adsorption of
metals onto the surface of a vessel, particularly in ultra-trace
1 This chapte,. has undergone phannacopoeial hannonisarion. See chapter
analyses. Contamination of sample solutions by metals and
5.8. Phannacopoeial harmonisation. Detennination oi M etal Catalyst 01'
M etal Reagem Residues
2014 Appendix VIII W V-A291
No Is the procedure described Yes

in the monograph?
Is lhe sample Is the sample

suitable for sui!able for
direct analysis? direc! analysis?
[5 the
compound No
Yes
soluble in
an aqueous
medium?
No
Is the
compound Yes
Yes soluble in
an organic Yes
medium?
Prepare the
No sample
solutions
Are the Perform

Yes
elemen!s dosed-vessel
volatile? digestion
No Perform open- or closed·

vessel digestion ar
incineration
No
Figure 2.4.20.-1. - Metal residues decision tree: sample preparation
ions present in the container can also lead to inaccurate limits of the metales) of interest. Analyse according to the
results. instructions of me manufacturer of the apparatus regarding
The use of volumetric glassware that does not comply with programme and wavelength.
Class A requirements of the appropriate Intemational System suitability A system suitability test must be
Standard of the Intemational Organization for carried out on the day of the analysis to ensure that the
Standardization (ISO) is acceptable if the validation or the sample preparation and measurement system are appropriate.
system suitability test of the method using such glassware Acceptance criterio n for preparation of sample solution '
have experimentally demonstrated that the method is suitable A clear solution is obtained.
for the intended purpose. Acceptance criterion for measurement system The
CAUTION: when using high-pressure digestion vessels and measured concentration of a standard solution of the metal
microwave laboratory equipment, the safety precautions and at a concentration within me range of the used calibration
operating instructions given by the manufacturer must be followed. curve do es not differ from me actual concentration by more
than 20 per cent.
Calculation The blank value of reagents must be taken
into account for me calculation of me contento Upon
completion of me analysis, me concentration of a given
Measurement metal in the sample is calculated by the software of the
Method The choice of the techniques depends mainly on instrument from the concentration of the metal in me test
the sample matrix and the characteristics and specification solution. If no calculation software is available or no
V-A292 Appendix VIII W 2014
No
Yes
No
Are the validation

requirements met?
Yes Is the recovery No

,-----<.of standard solution>-----------'
acceptable?
Yes
Figure 2.4.20.-2. - Metal residues decision tree: measurement
indication for caIculation is given in the corresponding demonstrated experimentally that such a procedure complies
general chapter in section 2.2. Physical and physicochemical with the validation requirements, with an appropriate system
methods, the concentration of a given metal in the sample can suitability test using material spiked with a suitable reference
be caIculated from the concentration of the metal in the material. The test materials must be spiked before any
solution using the following expression: sample preparation steps. For example, if a test material is to
e concentration of metal in the analysed sample, in be digested, the material must be spiked at the beginning of
micrograms per gram; the digestion procedure.
A instrument reading of the concentration of the metal Specificity
in the sample solution, in micrograms per millilitre; Specificity is the ability to ensure that the analytical
m mass of the sample in the initial sample solution, in procedures for sample preparation and measurement allow a
grams; reliable determination of the metales) in the presence of
VI volume of the initial sample preparation, in millilitres; components (e.g. carrier gas, impurities, matrix) that may be
V2 total volume of any dilution performed, in millilitres; expected to be presento
V3 volume of initial sample preparation used in any
dilution performed, in millilitres. Acceptance criteria The procedure must be able to
assess unequivocally each metal residue to be determined
with this procedure in the presence of components that may
Validation requirements
be expected to be present, including other metal residues,
Sorne validation requirements provided below may differ matrix components, and other sources of interference;
from those provided in general chapters of the Ph. Eur. specificity is demonstrated by complying with the accuracy
(e.g. 2.2.22 (AES), 2.2.23 (AAS), 2.2.57 (ICP-AES), 2.2.58 requirement for the metales) to be determined.
(ICP-MS)).
Range
Before the initial use of the selected procedure, the analyst
must ensure that the sample preparation and measurement Acceptance criterio n Range is demonstrated by
method are appropriate for the metales), sample matrix and complying with the recovery requirement.
instrument used. This is accomplished by following the Accuracy
validation procedure before the initial use and the system Verify the accuracy using a certified reference material
suitability test on the day of the analysis. (CRM) or by performing a test for recovery.
For metal residues, validation of a limit test must include Recovery Recovery may be determined on a sample of the
specificity and limit of detection. substance to be exarnined, spiked with a known quantity of a
The following section defines the characteristics for the reference standard of the metal (3 concentration levels in the
acceptability of a quantitative procedure. It must be range of 50-150 per cent of the intended specification limit,
2014 Appendix IX B V-A293
even if the original concentration of the reference standard is

at the specified value), in triplicate. Appendix IX
Acceptance criterion Spike recovery is within 70 per cent
and 150 per cent for the mean of 3 replicates at each
concentration.
A. Determination of Sulfated Ash
Use Method I unless otherwise directed.
Reapeatability
Test samples Either 6 independent samples of the Method 1
substance to be examined spiked with a suitable reference (No Ph. Eur. method)
standard at the specified concentration level, or 3 Heat a platinum dish to redness for 10 minutes, allow to cool
concentration levels prepared in triplicate. in a desiccator and weigh. Unless otherwise specified in the
Acceptance criterio n The relative standard deviation is in monograph, place 1 g of the substance being examined in the
both cases not more than 20 per cent. dish, moisten with sulfuric acid, ignite gently, again moisten
with sulfuric acid and ignite at about 800°. Cool, weigh again,
Intermediate precision
ignite for 15 minutes and repeat this procedure until two
The effect of random events (intra-laboratory variations) on successive weighings do not differ by more than 0.5 mg.
the analytical precision of the method must be established.
Acceptable experiments for establishing intermediate Method 11
precision include performing the repeatability analysis on (Ph. Eur. method 2.4.14)
different days, or with different instrumentation, or by Ignite a suitable crucible (for example, silica, platinum,
different analysts. Only 1 of the 3 experiments is required to porcelain or quartz) at 600 ± 50 oC for 30 min, allow to
demonstrate intermediate precision. cool in a desiccator over silica gel or other suitable desiccant
Acceptance criterio n The relative standard deviation is and weigh. Place the prescribed amount of the substance to
not more than 25 per cent. be examined in the crucible and weigh. Moisten the
Limit of quantification substance to be examined with a small amount of sulfuric
acid R (usually 1 mL) and heat gently at as low a
Determine the lowest concentration meeting the acceptance
temperature as practicable until the sample is thoroughly
criterion. Use the results from the accuracy study.
charred. After cooling, moisten the residue with a small
Acceptance criterion The limit of quantification is below amount of sulfuric acid R (usually 1 mL), heat gently until
the specification limito white fumes are no longer evolved and ignite at 600 ± 50 oC
Limit of detection (only applicable to limit tests) until the residue js completely incinerated. Ensure that fiames
Determine the lowest concentration giving a signal clearly are not produced at any time during the procedure. Allow
distinct from that obtained with a blank solution. the crucible to cool in a desiccator over silica gel or other
suitable desiccant, weigh it again and calculate the percentage
Acceptance criterio n The limit of detection is not more
of residue.
than 0.5 times the concentration of the specification limito
If the amount of the residue so obtained exceeds the
prescribed limit, repeat the moistening with sulfuric acid R
and ignition, as previously, for 30 min periods until 2
consecutive weighings do not differ by more than 0.5 mg or
until the percentage of residue complies with the prescribed
limito
The amount of substance used for the test (usually 1-2 g) is
chosen so that at the prescribed limit the mas s of the residue
(usuallyabout 1 mg) can be measured with sufficient
accuracy.
B. Determination of Sulfur Dioxide

Method 1
Apparatus A round-bottomed fiask of 1000- to 1500-rnL
capacity is fitted with a water-cooled refiux condenser the
upper end of which is connected to two absorption tubes in
series. The fiask is fitted with a gas inlet tube which reaches
nearly to the bottom of the fiask. Each absorption tube
contains 10 mL of hydrogen peroxide solution (20 voV
previously neutralised with O.IM sodium hydroxide VS using
bromophenol blue so/ution as indicator.
Method Place in the fiask 500 mL of water and 20 mL of
hydrochloric acid. Pass through the fiask a steady current of
nitrogen or carbon dioxide that has been bubbled through
dilute sodium carbonate solution and gradually heat the liquid
until it boils. Maintain the current of nitrogen or carbon
dioxide, allow the solution to boil for about 10 minutes and
cool the fiask by gradual irnrnersion in water. Introduce,
V-A294 Appendix IX e 2014
while momentarily removing the stopper of the flask, a of the test-tube with the aid of a liule water R to a 200 mL
weighed quantity of 50 to 100 g of the substance being wide-necked, conical flask. Heat on a water-bath for 15 min
examined, heat gently and boil for 45 minutes. Disconnect and allow to cool. Add 0.1 mL of a 1 gIL solution of
the absorption tubes before tuming off the current of nicrogen bromophenol blue R in ethanol (20 per cent V/V) R and titrate
or cm·bon dioxide and titrate the combined contents with with 0.1 M sodium hydraxide until the colour changes from
O.lM sodium hydroxide VS. Each mL of O.lM sodium hydroxide yellow to violet-blue (VI mL). Carry out a blank titration (V2
VS is equivalent to 3.203 mg of sulfur dioxide. mL).
Repeat the operation without the substance being examined. Calculate the content of sulfur dioxide in parts per million
The solution in the absorption tubes remains neutral. using the following expression:
n
32030 x (Vl - V2) x -
m
n molarity of the sodium hydroxide solution used as
titrant.
C. Determination of Water
Use Method lA unless otherwise directed.
Method 1
The semi-micro determination of water is based upon the
quantitative reaction of water with sulfur dioxide and iodine
in a suitable anhydrous medium in the presence of a base
with sufficient buffering capacity.
Apparatus The apparatus consists of a titration vessel
with:
- 2 identical platinum electrodes;
- tight inlets for introduction of solvent and titrant;
- an inlet for introduction of air via a desiccant;
- .a sample inlet fitted with a stopper or, for liquids, a
septum.
Inlet systems for introduction of dry nitro gen or for
aspiration of solvents may also be fitted.
The titration is carried out according to the instrument
supplier's instructions. Care is taken throughout the
determination to avoid exposure of reagents and solvents to
atmospheric moisture. The end-point is determined using 2
identical indicator electrodes connected to an electrical
source that maintains between the electro des either a
constant current (2.2.65. Valtamemc titratíon) or a constant
Figure 2.5.29.-1.- Apparatus for the determination of su/fur voltage (2.2.19. Amperometric titration). Where direct titration
dioxide is used (method A), addition of titrant causes either a
decrease in voltage where constant current is maintained or
Method II an increase in current where constant voltage is maintained,
until the end-point is reached. Instruments with automatic
end-point detection are commonly used.
Introduce 150 mL of water R into the flask (A) (see Figure
2.5 .29 .-1) and pass carbon dioxide R through the whole Standardisation To the titration vessel, add methanol R,
system for 15 min at arate of 100 ± 5 mUmin. To 10 mL dried if necessary, or the solvent recommended by the
of dilute hydrogen peroxide solution R add 0.15 mL of a 1 giL supplier of the titrant. Where applicable for the apparatus
solution of bromaphenol blue R in ethanol (20 per cent V/V) R. used, eliminate residual water from the measurement cell or
Add 0.1 M sodium hydraxide until a violet-blue colour is carry out a pre-titration. Introduce a suitable amount of
obtained, without exceeding the end-point. Place the solution water in an appropriate form (water R or a certified reference
in the test-tube (D) . Without interrupting the stream of material) and carry out the titration, stirring for the necessary
carbon dioxide, remove the funnel (B) and introduce through time. The water equivalent is not less than 80 per cent of
the opening into the flask (A) 25 .0 g (m g) of the substance that indicated by the supplier. Standardise the titrant before
to be examined with the aid of 100 mL of water R. Replace the first use and at suitable intervals thereafter.
the funnel. Close the tap of the funnel and add 80 mL of Unless otherwise prescribed, use Method A.
dilute hydrachloric acid R to the funnel. Open the tap of the Method A Introduce into the titration vessel methanal R,
funnel to allow the hydrochloric acid solution to flow into the or the solvent indicated in the monograph or recommended
flask, making sure that no sulfur dioxide escapes into the by the supplier of the titrant. Where applicable for the
funnel by c10sing the tap before the last few millilitres of apparatus used, eliminate residual water from the
hydrochloric acid solution drain out. Boil for 1 h. Open the measurement cell or carry out a pre-titration. Introduce the
tap of the funnel and stop the flow of carbon dioxide and substance to be examined rapidly and carry out the titration,
also the heating and the cooling water. Transfer the contents stirring for the necessary extraction time.
2014 Appendix IX e V-A295
Method B Introduce into the titration vessel methanol R, Method Clean the receiving tube and the condenser of the
or the solvent indicated in the monograph or recommended apparatus, thoroughly rinse with water, and dry.
by the supplier of the titrant. Where applicable for the Introduce 200 mL of toluene R and about 2 mL of water R
apparatus used, eliminate residual water from the into the dry flask. Distil for 2 h, then allow ro cool for about
measurement cell or carry out a pre-titration. Introduce the 30 min and read the water volume to the nearest 0.05 mL.
substance to be examined rapidly and in a suitable state of Place in the flask a quantity of the substance, weighed with
division. Add an accurately measured volume of the titrant, an accuracy of 1 per cent, expected ro give about 2 mL ro
sufficient to give an excess of about 1 mL or the prescribed 3 mL of water. If the substance has a pasty consistency,
volume. Allow ro stand protected from light for 1 min or the weigh it in a boat of metal foil. Add a few pieces of porous
prescribed time, with stirring. Titrate the excess of reagent material and heat the flask gently for 15 min. When the
using methanol R or the prescribed solvent, containing an roluene begins to boil, distil at the rate of about two drops
accurately known quantity of water. per second until most of the water has distilled over, then
Suitability The accuracy of the deterrnination with the increase the rate of distillation ro about four drops per
chosen titrant must be verified for each combination of second. When the water has all distilled over, rinse the inside
substance, titrant and solvent ro be examined. The following of the condenser tube with toluene R. Continue the
procedure, given as an example, is suitable for samples distillation for 5 min, remove the heat, allow the receiving
containing 2.5-25 mg of water. tube ro cool to room temperature and dislodge any droplets
The water content of the substance ro be examined is of water which adhere ro the walls of the receiving tube.
deterrnined using the reagent/solvent system chosen. When the water and roluene have completely separated, read
Thereafter, in the same titration vessel, sequential known the volume of water and calculate the content present in the
amounts of water, corresponding ro about 50-100 per cent of substance as millilitres per kilogram, using the formula :
the amount found in the substance to be examined, are
added in an appropriate form (at least 5 additions) and the 1000 (n2 - nl)
water content is deterrnined after each addition. Calculate m
the percentage recovery (r) after each addition using the
following expression: m the mass in grams of the substance ro be examined,
n¡ the number of millilitres of water obtained in the first
r = 100 W 2
distillation,
W1
n2 the total number of millilitres of water obtained in the
W¡ = amount of water added, in milligrams; 2 distillations.
W2 = amount of water found, in milligrams.
Calculate the mean percentage recovery ( r ).
The reagent/solvent system is considered to be acceptable if r
is between 97.5 per cent and 102.5 per cent.
Calculate the regression line. The x-axis represents the
cumulative water added whereas the y-axis represents the
sum of the initial water content deterrnined for the substance o
o
(M) and the cumulative water deterrnined after each N
1\1
addition. Calculate the slope (b), the intercept with the y-axis e
(a) and the intercept of the extrapolated calibration line with
the x-axis (d).
Calculate the percentage errors (el and e2) using the
following expressions:
a the y-axis intercept, in milligrams of water;

d the x-axis intercept, in milligrams of water;
M water content of the substance, in milligrams of water. E
The reagentlsolvent system is considered ro be acceptable if:

- je¡f and je2j and are not greater than 2.5 per cent;
- b is between 0.975 and 1.025.
Method 11 Determination of water by distillation

(Ph. Eur. method 2.2. 13)
The apparatus (see Figure 2.2. 13.-1) consists of a glass flask
(A) connected by a tube (D) ro a cylindrical tube (B) fitted
with a graduated receiving tube (E) and reflux condenser
(C). The receiving tube (E) is graduated in 0.1 mL. Figure 2.2.13.-1. - Apparatus for the determination ofwater
The source of heat is preferably an electric heater with by distillation
rheostat control or an oil bath. The upper portion of the flask
and the connecting tube may be insulated.
V-A296 Appendix IX D 2014
Method III (Coulometric titration) Verification ofthe accuracy Between two successive
(Ph. Eur. method 2.5.32) sample titrations, introduce an accurately weighed amount of
PrincipIe The coulometric titration of water is based upon water in the same order of magnitude as the amount of
the quantitative reaction of water with sulfur dioxide and water in the sample, either as water R or in the fonn of
iodine in an anhydrous medium in the presence of a base standard solution for the micro determination oi water R, and
with sufficient buffering capacity. In contrast to the perfonn the coulometric titration. The recovery rate is within
volumetric method described under (2.5.12), iodine is the range from 97.5 per cent to 102.5 per cent for an
produced electrochemically in the reaction cel! by oxidation addition of 1000 ¡.¡g of H 2 0 and in the range from
of iodide. The iodine produced at the ano de reacts 90.0 per cent to 110.0 per cent for the addition of 100 ¡.¡g of
immediately with the water and the sulfur dioxide contained H 2 0.
in the reaction cell. The amount of water in the substance is
directly proportional to the quantity of electricity up until the
titration end-point. When all of the water in the cell has
been consumed, the end-point is reached and thus an excess D. Determination of Loss on Drying
of iodine appears. 1 mole of iodine corresponds to 1 mole of (Ph. Eu/'. method 2.2.32)
water, a quantity of electricity of 10.71 C corresponds to Loss on drying is the loss of mass expressed as per cent mini.
1 mg of water. Method Place the prescribed quantity of the substance to
Moistute is eliminated from the system by pre-electrolysis. be examined in a weighing bottle previously dried under the
Individual detenninations can be carried out successively in conditions prescribed for the substance to be examined.
the same reagent solution, under the following conditions: Dry the substance to constant mass or for the prescribed
- each component of the test mixture is compatible with the time by one of the following procedures. Where the drying
other components, temperature is indicated by a single value rather than a
- no other reactions take place, range, drying is carried out at the prescribed temperature
± 2 oc.
- the volume and the water capacity of the electrolyte
reagent are sufficient. a) "in a desiccator": the drying is carried out over
diphosphorus pentoxide R at atmospheric pressure and at
Coulometric titration is restricted to the quantitative
room temperature;
detennination of small amounts of water, a range of 10 ¡.¡g up
to 10 mg of water is recommended. b) "in vacuo": the drying is carried out over diphosphonls
pentoxide R, at a pressure of 1.5 kPa to 2.5 kPa at room
Accuracy and precision of the method are predominantly
temperature;
governed by the extent to which atmospheric moisture is
excluded from the system. Control of the system must be c) "in vacuo within a specified temperature range": the
monitored by measuring the amount of baseline drift. drying is carried out over diphosphorns pentoxide R, at a
pressure of 1.5 kPa to 2.5 kPa within the temperature
Apparatus The apparatus consists of a reaction cell,
range prescribed in the monograph;
electrodes and magnetic stirrer. The reaction cell consists of
a large anode compartment and a smaller cathode d) "in an oven within a specified temperature range": the
compartment. Depending on the design of the electrode, drying is carried out in an oven within the temperature
both compartments can be separated by a diaphragm. Each range prescribed in the monograph;
compartment contains a platinum electrode. Liquid or e) "under high vacuum": the drying is carried out over
solubilised samples are introduced through a septum, using a diphosphonls pentoxide R at a pressure not exceeding 0.1
syringe. Alternatively, an evaporation technique may be used kPa, at the temperature prescribed in the monograph.
in which the sample is heated in a tube (oven) and the water If other conditions are prescribed, the procedure to be used
is evaporated and carried into the cell by means of a stream is described in ful! in the monograph.
of dry inert gas. The introduction of solid samples into the
cell should in general be avoided. However, if it has to be
done it is effected through a sealable port; appropriate
precautions must be taken to avoid the introduction of E. Limit Test for Carbon Monoxide in
moisture from air, such as working in a glove box in an
atmosphere of dry inert gas. The analytical procedure is Medicinal Gases
controlled by a suitable electronic device, which also displays (Ph. Eur. method 2.5.25)
the results.
Method Fill the compartments of the reaction cell with METHODI
electrolyte reagent for the micro determinatwn of water R Apparatus The apparatus (Figure 2.5 .25.-1) consists of
according to the manufacturer's instructions and perfonn the the following parts connected in series:
coulometric titration to a stable end-point. Introduce the - a U-tube (U¡) containing anhydrous silica gel R
prescribed amount of the substance to be examined into the impregnated with chl'Omium trioxide R;
reaction cell, stir for 30 s, if not otherwise indicated in the
- a wash bottle (F¡) containing 100 mL of a 400 gIL
monograph, and titrate again to a stable end-point. In case solution of potassium hydroxide R;
an oven is used, the prescribed sample amount is introduced
into the tube and heated. After evaporation of the water - a U-tube (U2 ) containing pellets of potassium hydroxide R;
from the sample into the titration cell, the titration is started. - a U-tube (U3 ) containing diphosphorns pentoxide R
Read the value from the instrurnent's output and calculate if dispersed on previously granulated, fused pumice;
necessary the percentage or amount of water that is present - a U-tube (U4 ) containing 30 g of recrystallised iodine
in the substance. When appropriate to the type of sample pentoxide R in granules, previously dried at 200 oC and
and the sample preparation, perform a blank titration. kept at a temperature of 120 oC (7) during the test;
the iodine pentoxide is packed in the rube in 1 cm
2014 Appendix IX F V-A297
U1 F1 U2 U3 U4 F2
8..-
1-
- 1 1--
-: 1 1-_
100m
:1 i--
-o:
- 1 1-
1
':-1 1-
_ ,1-
Figure 2.5.25.-1. - Apparatus for the determination of carbon monoxide

columns separated by 1 cm columns of glass wool to give concentration. To prevent the entry of particles into the
an effective length of 5 cm; sensors, which could cause stray-light phenomena, the
- a reaction tu be (F2) containing 2.0 mL of pocassium iodide apparatus is fitted with a suitable filter.
solution R and 0.15 mL of starch solution R. Required technical specifications When used for a limit
Method Flush the apparatus with 5.0 L of argon R and, if test, the carbon monoxide infrared analyser meets the
necessary, discharge the bll.!e colour in the iodide solution by foHowing technical specifications:
adding the smallest necessary quantity of freshly prepared - limit 01 detection: (generally defined as a signal-to-noise
0.002 M sodium thiosulfate. Continue fiushing until not more ratio of 2) maximum 20 per cent of the maximum
than 0.045 mL of 0.002 M sodium thiosulfate is required after admissible concentration;
passage of 5.0 L of argon R. Pass the gas to be examined - repeatabiliry: maximum relative standard deviation of
from the cylinder through the apparatus, using the prescribed 10 per cent of the maximum admissible concentration,
volume and the fiow rateo Flush the last traces of liberated determined on 6 measurements;
iodine into the reaction tube by passing through the - linearity: maximum ·l O per cent of the maximum
apparatus 1.0 L of argon R. Titrate the liberated iodine with admissible concentration.
0.002 M sodium thiosulfate. Carry out a blank test, using the
prescribed volume of argon R . The difference between the The technical specifications must be met in the presence of
volumes of 0.002 M sodium thiosulfate used in the titrations is the other gas impurities in the sample.
not greater than the prescribed limit.
METHOD 11 F. Determination of Carbon Dioxide in

Gases absorb light at one or more specific wavelengths. This
property is widely used to allow highly selective measurement
Medicinal Gases
of their concentrations. (Ph. Eur. method 2.5.24)
Description and principIe of measurement The Gases absorb light at one or more specific wavelengths. This
concentration of carbon monoxide in other gases can be property is widely used to allow highly selective measurement
determined using an inftared analyser. of their concentrations.
The infrared analyser gene rally consists of a light source Description and principIe of measurement. The
emitting broadband infrared radiation, an optical device, a concentration of carbon dioxide in other gases can be
sample cell and a detector. The optical device may be determined using an infrared analyser.
positioned either before or after the sample cell; it consists of The infrared analyser generally consists of a light source
one or several optical filters, through which the broadband emitting broadband infrared radiation, an optical device, a
radiation is passed. The optical device in this case is selected sample ceH and a detector. The optical device may be
for carbon monoxide. The measurement light beam passes positioned either before or after the sample ceH and it
through the sample cell and may also pass through a consists of one or several optica! filters, through which the
reference ceIl if the analyser integrates such a feature (sorne broadband radiation is passed. The optical device in this case
use an electronic system instead of a reference cel!) . is selected for carbon dioxide. The measurement light beam
When carbon monoxide is present in the sample cell, passes through the sample ceH and may also pass through a
absorption of energy in the measurement light beam wilI reference ceH if the analyser integra tes such a feature (sorne
occur according to the Beer-Lambert law and this produces a use an electronic system instead of a reference cel!).
change in the detector signa!. This measurement signal is When carbon dioxide is present in the sample cell,
compared to a reference signal to generate an output related absorption of energy in the measurement light beam will
to the concentration of carbon monoxide. The generated occur according to the Beer-Lambert law and this produces a
signal is linearised in order to obtain the carbon monoxide change in the detector signa!. This measurement signal is
V-A298 Appendix IX G 2014
compared to a reference signal to generate an output related - a chamber in which nitro gen monoxide and ozone can
to the concentration of carbon dioxide. The generated signal react,
is linearised in order to obtain the carbon di oxide - a system for detecting light radiation emitted at a
concentration. To prevent the entry of particles into the wavelength of 1.2 ¡.tm, consisting of a selective optical
sensors, which could cause stray-light phenomena, the filter and a photomultiplier tube.
apparatus is fitted with a suitable filter.
Required technical specifications When used for a limit
test, the infrared analyser meets the following technical H. Determination of Oxygen in Medicinal
specifications:
- limit 01 detection: (generally defined as a signal-to-noise Gases
ratio of 2) maximum 20 per cent of the maximum (Ph. Eur. methad 2.5.27)
admissible concentration; Oxygen in gases is determined using a paramagnetic analyser.
- repeatability: maximum relative standard deviation of The principIe of the method is based on the high
10 per cent of the maximum admissible concentration, paramagnetic sensitivity of the oxygen molecule. Oxygen
determined on 6 measurements; exerts a strong interaction on magnetic fields, which is
- linearity: maximum 10 per cent of the maximum measured electronically, amplified and converted to a reading
admissible concentration. of oxygen concentration. The measurement of oxygen
The technical specifications must be met in the presence of concentration is dependent upon the pressure and
the other gas impurities in the sample. temperature and, if the analyser is not automatically
compensated for variations in temperature and pressure, it
must be calibrated irnmediately prior to use. As the
paramagnetic effect of oxygen is linear, the instrument must
G. Determination of Nitrogen Monoxide have a suitable range with a readability of 0.1 per cent or
better.
and Nitrogen Dioxide in Medicinal Gases Calibration 01 the instrument Make the setting in the
(Ph. Eur. method 2.5.26) following manner:
Nitrogen monoxide and nitro gen dioxide in gases are - set the zero by passing nitrogen RI through the instrument
determined using a chemiluminescence analyser (Figure until a constant reading is obtained;
2.5.26.-1). - set the scale to 100 per cent by passing oxygen R through
The apparatus consists of the following: the instrument at the same flow rate as for nitrogen RI
- a device for filtering, checking and controlling the flow of until a constant reading is obtained.
the gas to be examined, Assay Pass the gas to be examined through the instrument
- a converter that reduces nitro gen dioxide to nitrogen at a constant flow rate until a constant reading is obtained.
monoxide, to determine the combined content of nitrogen Record the concentration of oxygen in the gas to be
monoxide and nitrogen dioxide. The efficiency of the examined.
converter has to be verified prior to use,
- a controlled-flow-rate ozone generator; the ozone is
produced by high-voltage electric discharges across two J. Determination of Water in Medicinal
electrodes; the ozone generator is supplied with pure
oxygen or with dehydrated ambient air and the
Gases
concentration of ozone obtained must greatly exceed the (Ph. Eur. method 2.5.28)
maximum content of any detectable nitrogen oxides, Water in gases is determined using an electrolytic
hygrometer, described below.
Sample flow control
Reaction chamber
Converter
N02-NO / optical filter .
Refrigerated chamber
Filter to eliminate =t~at:1 Photomulti-

~- ozone plier tube
Ozone generator
system
- Controls
- NO - (NO+N02) cycle
NO (NO+N02) N02
Figure 2.5.26.-1. - Chemiluminescence analyser

2014 Appendix IX L V-A299
2 3 4
The measuring cell consists of a thin film of diphosphorus
pentoxide, between 2 coiled platinum wires which act as r--------,---------------- 7
electrodes. The water vapour in the gas to be examined is
absorbed by the diphosphorus pentoxide, which is
transformed to phosphoric acid, an electrical conductor. 5
A continuous voltage applied across the electrodes produces
electrolysis of the water and the regeneration of the
diphosphorus pentoxide. The resulting electric current, which
is proportional to the water content in the gas to be 6
examined, is measured. This system is self-calibrating since it

obeys Faraday's law.
Take a sample of the gas to be examined. Allow the gas to 1. Gas supply 5. Indicator tube
stabilise at room temperature. Purge the cell continuously
2. Pressure regulator 6. Indicator tube pump
until a stable reading is obtained. Measure the water content
in the gas to be examined, making sure that the temperature 3. Needle valve 7. End open to atmosphere
is constant throughout the device used to introduce the gas 4. "Y"-piece
into the apparatus .
Figure 2.1.6.-1. - Apparatus for gas detector tubes
Carbon dioxide detector tube Sealed glass tube

K. Gas Detector Tubes containing adsorbent filters and suitable supports for
(Ph. Eur. text 2.1.6) hydrazine and crystal violet indicators. The minimum value
Gas detector tubes are cylindrical, sealed tubes consisting of indicated is 100 ppm with a relative standard deviation of at
an inert transparent material and are constructed to allow the most ± 15 per cent.
passage of gas. They contain reagents adsorbed onto inert Sulfur dioxide detector tube Sealed glass tube containing
substrates that are suitable for the visualisation of the adsorbent filters and suitable supports for the iodine and
substance to be detected and, if necessary, they also contain starch indicator. The minimum value indicated is 0.5 ppm
preliminary layers and/or adsorbent filters to elimina te with a relative standard deviation of at most ± 15 per cent.
substances that interfere with the substance to be detected. Di! detector tube Sealed glass tube containing adsorbent
The layer of indicator contains either a single reagent for the filters and suitable supports for the sulfuric acid indicator.
detection of a given impurity or several reagents for the The minimum value indicated is 0.1 mglm3 with a.relative
detection of several substances (monolayer tube or multilayer standard deviation of at most ± 30 per cent.
tube). Nitrogen monoxide and nitrogen dioxide detector tube
The test is carried out by passing the required volume of the Sealed glass tube containing adsorbent filters and suitable
gas to be examined through the indicator tube. The length of supports for an oxidising layer (Cr(VI) salt) and the
the coloured layer or the intensity of a colour change on a diphenylbenzidine indicator. The minimum value indicated is
graduated scale gives an indication of the impurities presento 0.5 ppm with a relative standard deviation of at most
The calibration of the detector tubes is verified according to ± 15 per cent.
the manufacturer's instructions. Carbon monoxide detector tube Sealed glass tube
Operating conditions Examine according to the containing adsorbent filters and suitable supports for
manufacturer's instructions or proceed as follows : di-iodine pentoxide, se1enium dioxide and fuming sulfuric
The gas supply is connected to a suitable pressure regulator acid indicators. The minimum value indicated is 5 ppm or
and needle valve. Connect the flexible tubing fitted with a less, with a relative standard deviation of at most
y -piece to the valve and adjust the flow of gas to be . ± 15 per cent.
examined to purge the tubing in order to obtain an Hydrogen sulfide detector tube Sealed glass tube
appropriate flow (Figure ;U.6.-1). Prepare the indicator tube containing adsorbent filters and suitable supports for an
and fit to the metering pump, following the manufacturer's appropriate lead salt indicator. The minimum value indicated
instructions. Connect the open end of the indicator tube to is 1 ppm or less, with a relative standard deviation of at most
the short leg of the tubing and operate the pump by the ± 10 per cent.
appropriate number of strokes to pass a suitable volume of Water vapour detector tube Sealed glass tube containing
gas to be examined through the tube. Read the value adsorbent filters and suitable supports for the magnesium
corresponding to the length of the coloured layer or the perchlorate indicator. The minimum value indicated is
intensity of the colour on the graduated scale. If a negative 67 ppm or less, with a relative standard deviation of at most
result is achieved, indicator tubes can be verified with a ± 20 per cent.
calibration gas containing the appropriate impurity.
In view of the wide variety of available compressor oils, it is
necessary to verify the reactivity of the oil detector tubes for
the oil used. Information on the reactivity for various oils is L. Determination of Nitrous Oxide in
given in the leaflet supplied with the tube. If the oil used is
not cited in the leaflet, the tube manufacturer must verify the
Gases
reactivity and if necessary provide a tube specific for this oil. (Ph. Eur. method 2.5.35)
Gases absorb light at one or more specific wavelengths. This
property is widely used to allow highly selective measurement
of their concentrations.
V -A300 Appendix IX M 2014
Description and principie of measurement The - specific surface area of the solid, as well as such properties
concentration of nitrous oxide in other gases can be as degree of crystallinity, degree of porosity, and glass
determined using an infrared analyser. transition and melting temperature;
The infrared analyser generally consists of a light source - site of water interaction, the extent of binding, and the
emitting broadband infrared radiation, an optical device, a degree of molecular mobility;
sample cell and a detector. The optical device may be - effects of temperature and relative humidity;
positioned either before or after the sample cell and it - essentially irreversible hydration;
consists of one or several optical filters, through which the
broadband radiation is passed. The optical device in this case - kinetics of moisture uptake;
is selected for nitrous oxide. The measurement light beam - various factors that might infiuence the rate at which
passes through the sample cell and may also pass through a water vapour can be taken up by a solid;
reference cell if the analyser integrates such a feature (sorne - for water-soluble solids capable of being dissolved by the
use an electronic system instead of a reference cell). sorbed water, under which conditions dissolution will take
When nitrous oxide is present in the sample cell, absorption place.
of energy in the measurement light beam will occur
according to the Beer-Lambert law and this produces a PHYSICAL STATES OF SORBED WATER
change in the detector signa!. This measurement signal is Water can physically interact with solids in different ways.
compared to a reference signal to generate an output related It can interact at the surface (adsorption) or it can penetrate
to the concentration of nitrous oxide. The generated signal is the bulk solid structure (absorption). When both adsorption
linearised in order to obtain the nitrous oxide concentration. and absorption occur, the term sorption is often used.
To prevent the entry of partieles into the sensors, which Adsorption is particularly critical in affecting the properties of
could cause stray-light phenomena, the apparatus is fitted solids when the specific surface area is large. Large values of
with a suitable filter. specific surface area are seen with solids having very small
partieles, as well as with solids having a high degree of
intrapartiele porosity. Absorption is characterised by an
association of water per gram of solid that is much greater
M. Water-So lid Interactions: than that which can form a mono molecular layer on the
availab1e surface, and an amount that is generally
Determination of Sorption-Desorption independent of the specific surface area.
Isotherms and of Water Activity Most crystalline solids will not absorb water into their bulk
structures because of the elose packing and high degree of
order of the crystal lattice. Indeed, it has been shown that the
INTRODUCTION degree of absorption into solids exhibiting partíal crystallinity
and partial amorphous structure is often inversely
Pharmaceutical solids as raw materials or as constituents of proportional to the degree of crystallinity. With sorne
dosage forms most often come in contact with water during crystalline solids, however, crystal hydrates may formo These
processing and storage. This may occur (a) during
hydrates may exhibit a stoichiometric "relationship, in terms of
crystallisation, lyophilisation, wet granulation, or spray
water molecules bound per solid molecule, or they may be
drying; and (b) because of exposure upon handling and
non-stoichiometric. Upon dehydration, crystal hydrates may
storage to an atrnosphere containing water vapour or
either retain their original crystal structure, or lose their
exposure to other material s in a dosage form that contain crystallinity and become amorphous, or transform into a new
water capable of distributing it to other ingredients. Sorne
anhydrous or less-hydrated crystal form o
properties known to be altered by the association of solids
with water inelude rates of chemical degradation in the Amorphous or partially amorphous solids are capable of
"solid-state", crystal growth and dissolution, dispersibility taking up significant amounts of water because there is
and wetting, powder fiow, lubricity, powder compactibility, sufficient molecular disorder in the solid to permit
compact hardness and microbial contamination. penetration, swelling or dissolution. Such behaviour is
observed with most amorphous polymers and with small-
Although precautions can be takeu when water is perceived
molecular-mass solids rendered. amorphous during
to be a problem, i.e. eliminating all moisture, reducing
preparation, e.g. by lyophilisation, or after milling.
contact with the atrnosphere, or controlling the re!ative
The introduction of defects into highly crystalline solids will
humidity of the atrnosphere, such precautions generally add
also produce this behaviour. The greater the chemical affinity
expense to the process with no guarantee that during the life
of water for the solid, the greater the total amount that can
of the product further problems associated with moisture will
be absorbed. When water is absorbed by amorphous solids,
be avoided. It is also important to recognise that there are
the bulk properties of the solid can be significantly altered.
many situations where a certain leve! of water in a solid is
It is well established, for example, that amorphous solids,
required for proper performance, e.g. powder compaction.
depending on the temperature, can exist in at least one of
It is essential for both reasons, therefore, that as much as
2 states, "glassy" or "fluid"; the temperature at which one
possible is known about the effects of moisture on solids
state transforms iuto the other is the glass transition
before strategies are developed for their handling, storage and
temperature, Tg •
use.
Water absorbed into the bulk solid structure, by virtue of its
Sorne of the more critical pieces of required information
effect on the free volume of the solid, can act as an efficient
conceming water-solid interactions are:
plasticiser and reduce the value of Tg . Since the rheological
- total amounl of water present; properties of "fluid" and "glassy" states are quite different,
- the extent to which adsorption and absorption occur; i.e. the "fluid" state exhibits much les s viscosity as one goes
- whether or not hydrates form; increasingly aboye the glass transition temperature, it is not
surprising that a number of important bulk properties
2014 Appendix IX M V-A301
dependent on the rheology of the solid are affected by via deliquescence and continue to dissolve over a long time
moisture contento Since amorphous solids are metastable periodo The rate of water uptake in general depends on a
relative to the crystalline form of the material, with small- number of parameters not found to be critical in equilibrium
molecular-mas s materials, it is possible for absorbed moisture measurements because rates of sorption are primarily mas s-
to initiate reversion of the solid to the crystalline form, transfer controlled with sorne contributions from heat-
particularly if the solid is transformed by the sorbed water to transfer mechanisms. Thus, factors such as vapour diffusion
a "fluid" state. This is the basis of "cake collapse" often coefficients in air and in the solid, convective airflow, and
observed during the lyophilisation process. An additional the surface area and geometry of the solid bed and
phenomenon noted specifically with water-soluble solids is surrounding environment, can play an important role.
their tendency to deliquesce, i.e. to dissolve in their own Indeed, the method used to make measurements can often
sorbed water, at relative humidities, RH¡, in excess of the be the rate-determining factor because of these
relative humidity of a saturated solution of the solid, RHo. environmental and geometric factors .
Deliquescence arises because of the high water solubility of
the solid and the significant effect it has on the colligative DETERMINATION OF SORPTION-DESORPTION
properties of water. lt is a dynamic process that continues to ISOTHERMS
occur as long as RH¡ is greater than RHo. PrincipIe The tendency to take up water vapour is best
The key to understanding the effects water can have on the assessed by measuring sorption or desorption as a function of
properties of solids, and vice versa, rests with an relative humidity, at constant temperature, and under
understanding of the location of the water molecule and its conditions where sorption or desorption is essentially
physical state. More specifically, water associated with solids occurring independently of time, i.e. equilibrium. Relative
can exist in a state that is directly bound to the solid, as well humidity, RH, is defined by the following expression:
as in a state of mobility approaching that of bulk water. This
difference in mobility has been observed through such Pe X 100
measurements as heats of sorption, freezing point, nuclear Po
magnetic resonance, dielectric properties and diffusion. Such
changes in mobility have been interpreted as arising because Pe pressure of water vapour in the system;
of changes in the thermodynamic state ofwater as more and Po saturation pressure of water vapour under the same
more water is sorbed. Thus, water bound directly to a solid conditions.
is often thought as unavailable to affect the properties of the The ratio Pj Po is referred to as the relative pressure.
solid, whereas larger amounts of sorbed water may become Sorption or water uptake is best assessed starting with dried
more clustered and form water more like that exhibiting samples and subjecting them to a known relative humidity.
solvent properties. In the case of crystal hydrates, the Desorption is studied by beginning with a system already
combination of intermolecular forces (hydrogen bonding) containing sorbed water and reducing the relative humidity.
and crystal packing can produce very strong water-solid As the name indica tes, the sorption-desorption isotherm is
interactions. Recognising that the presence of water in an valid only for the reference temperature, hence a special
amorphous solid can affect the glass transition temperature isotherm exists for each temperature. Ordinarily, at
and hence the physical state of the solid, at low levels of equilibrium, moisture content at a particular relative
water, most polar amorphous solids are in a highly viscous humidity must be the same, whether determined from
glassy state because of their high values of Tg . Hence, water sorption or desorption measurements. However, it is
is "frozen" into the solid structure and is rendered immobile common to see sorption-desorption hysteresis.
by the high viscosity, e.g. 10 13 Pa·s . As the amount ofwater
Methods Samples may be stored in chambers at various
sorbed increases and Tg de creases, approaching ambient
relative humidities (Figure 2.9.39.-1) . The mas s gained or
temperatures, the glassy state approaches that of a "fluid"
lost for each sample is then measured. The major advantage
state and water mobility along with the mobility of the solid
of this method is convenience, while the major disadvantages
itself increases significantly. At high RH, the degree of water
are the slow 'rate of reaching constant mass, particularly at
plasticisation of the solid can be sufficiently high so that
high relative humidities, and the error introduced in opening
water and the solid can now achieve significant amounts of
and closing the chamber for weighing.
mobility. In general, therefore, this picture of the nature of
sorbed water helps to explain the rather significant effect Dynamic gravimetric water sorption systems allow the on-line
moisture can have on a number of bulk properties of solids weighing of a sample in a controlled system to assess the
such as chemical reactivity and mechanical deformation. interaction of the material with moisture at various
lt suggests strongly that methods of evaluating chemical and prograrnmable levels of relative humidity at a constant
physical stability of solids and solid dosage forms take into temperature. The major benefit of a controlled system is that
account the effects water can have on the solid when it is isothermal conditions cán be more reliably established and
sorbed, particularly when it enters the solid structure and that the dynamic response of the sample to changing
acts as a plasticiser. conditions can be monitored. Data points for the
determination of the sorption isotherm (e.g. from O per cent
Rates of water uptake The rate and extent to which
to approximately 95 per cent RH, non condensing) are only
solids exposed to the atmosphere might either sorb or desorb
taken after a sufficiently constant signal indicates that the
water vapour can be a critical factor in the handling of
sample has reached equilibrium at a given level of humidity.
solids. Even the simple act of weighing out samples of solid
In sorne cases (e.g. deliquescence), the maximum time may
on an analytical balance. and the exposure, therefore, of a
be restricted although the equilibrium level is not reached.
thin layer of powder to the atmosphere for a few minutes
The apparatus must adequately control the temperature to
can lead to significant error in, for example, the estimation of
ensure a good baseline stability as well as accurate control of
loss on drying values. lt is well established that water-soluble
the relative humidity generation. The required relative
solids exposed to relative humidities aboye that exhibited by
humidities can be generated, e.g. by accurately mixing dry
a saturated solution of that solid will spontaneously dissolve
and saturated vapour gas with flow controllers.
V-A302 Appendix IX M 2014
A B
1 D
L
H G F
A. Humidity controller D. Humidity regulated module G. Vapour humidifier
B. Temperature controlled chamber E. Reference H. Flow control module
C. Balance module F. Sample 1. Dry gas
Figure 2.9.39.-1. - Example of an apparatus for the determination ofthe water sorption (other designsare possible)
The electrostaúc behaviour of the powder must also be Adsorption-desorption hysteresis can be interpreted, for
considered. The verificaúon of the temperature and the example, in terms of the porosity of the sample, its state of
relaúve humidity (controlled with, for example, a certified agglomeration (capillary condensation), the formation of
hygrometer, certified salt solutions or deliquescence points of hydrates, polymorphic change, or liquefying of the sample.
cerúfied salts over an adequate range), must be consistent Certain types of systems, particularly those with microporous
with the instrument specification. The balance must provide solids and amorphous solids, are capable of sorbing large
a sufficient mas s resolution and long term stability. amounts of water vapour. Here, the amount of water
It is also possible to measure amounts of water uptake not associated with the solid as relative humidity is decreased, is
detectable gravimetrically using volumetric techniques. greater than the amount that originally sorbed as the relaúve
In sorne cases, direct analysis of water content by different humidity was increased. For microporous solids, vapour
methods such as determination of the boiling point, adsorption-desorption hysteresis is an equilibrium
determinaúon of water by distillation, loss on drying or gas phenomenon associated with the process of capillary
chromatography may be advantageous. In the case of condensatión. This takes place because of the high degree of
adsorption, to improve sensiúvity, one can increase the irregular curvature of the micropores and the fact that they
specific surface area of the sample by reducing particle size or "fill" (adsorption) and " empty" (desorption) under different
by using larger samples to increase the total area. It is equilibrium conditions. For non-porous solids capable of
important, however, that such comminuúon of the solid do es absorbing water, hysteresis occurs because of a change in the
not alter the surface structure of the solid or render it more degree of vapour-solid interaction due to a change in the
amorphous or otherwise less ordered in crystallinity. equilibrium state of the solid, e.g. conformation of polymer
For absorpúon, where water uptake is independent of specific chains, or because the time scale for structural equilibrium is
surface area, only increasing sample size will help. Increasing longer than the time scale for water desorption. In measuring
sample size, however, will increase the time to establish sorne sorption-desorpúon isotherms, it is therefore important to
type of equilibrium. To establish accurate values, it is establish that something close to an equilibrium state has
important to get desolvation of the sample as thoroughly as been reached. Particularly with hydrophilic polymers at high
possible. Higher temperatures and lower pressures (vacuum) relative humidities, the establishment of water sorption or
facilita te this process; however, one must be aware of any desorption values independent of úme is quite difficult, since
adverse effecís this might have on the solid such as one is usually dealing with a polymer plasticised into its
dehydration, chemical degradaúon or sublimaúon. Using "fluid" sta te, where the solid is undergoing significant
higher temperatures to induce desorpúon, as in a change.
thermogravimetric apparatus, likewise must be carefully In the case of crystal hydrate formation, the plot of water
carried out beca use of these possible pitfalls. uptake versus pressure or relative humidity will in these cases
Report and interpretation of the data Sorpúon data are exhibit a sharp increase in uptake at a particular pressure and
usually reported as a graph of the apparent mas s change in the amount of water taken up will usually exhibit a
per cent of the mass of the dry sample as a function of stoichiometric mole:mole ratio of water to solido In sorne
relative humidity or time. Sorption isotherms are reported cases, however, crystal hydrates will not appear to undergo a
both in tabular form and as a graph. The measurement phase change or the anhydrous form will appear amorphous.
method must be traceable with the data. Consequently, water sorption or desorption may appear more
2014 Appendix IX M V-A303
like that seen with adsorption processes. X-ray begins is the dew point from which the ERH is determined.
crystalIographic analysis and thermal analysis are particularly Commercially available instruments using the dew
useful for the study of such systems . pointlchilled mirror method or other technologies need to be
For situations where water vapour adsorption occurs evaluated for suitability, qualified, and calibrated when used
predominantly, it is very helpful to measure the specific to make water activity determinations. These instruments are
surface area of the solid by an independent method and to typically calibrated over an adequate range, for example,
express adsorption as mas s of water sorbed per unit area of using sorne saturated salt solutions at 25 cC such as those
solid surface. This can be very useful in assessing the possible listed in Table 2.9.39 .-1.
importance of water sorption in afIecting solid properties.
For example, 0.5 per cent m /m uptake of water could hardly Table 2.9.39.-1. - Standard saturated salt solutions
cover the bare surface of 100 m 2(g, while for 1.0 m 2/g this Saluraled salts solulions ERH
al 25 oc
Aw
amounts to 100 times more surface coverage. In the case of (per cenl)
pharmaceutical solids which have a specific surface area in Potassium sulfate
the range of 0.01 m 2/g to 10 m 2/g, what appears to be low (K,SO.)
97.3 0.973
water content could represent a significant amount of water

Barium chloride
for the available surface. Since the "dry surface area" is not a 90.2 0.902
(BaCI 2)
fa<;:tor in absorption, sorption of water with amorphous or
partially amorphous solids can be expressed on the basis of Sodium chloride
75.3 0.753
unit mas s corrected for crystallinity, when the crystal form (NaCl)
do es not sorb significant amounts of water relative to the Magnesium nitrate

52.9 0.529
amorphous regions. (Mg(N0 3) , )
Magnesium chloride
DETERMINATION OF THE WATER ACTIVITY (MgCI,)
32.8 0.328
PrincipIe Water activity, A w , is the ratio of vapour Lithium chloride

pressure of water in the product (P) to saturation pressure of (LiCI)
11.2 0.112
water vapour (Po) at the same temperature. It is numerically
equal to 1/100 of the relative humidity (RH) generated by
the product in a closed system. RH can be ca1culated from
direct measurements of partial vapour pressure or dew point,
or from indirect measurement by sensors whose physical or
electric characteristics are altered by the RH to which they
are exposed. Ignoring activity coefficients, the relationship
between A w and equilibrium relative humidity (ERH) are
represented by the following equations:
P
Aw:= -
Po
ERH (per cent) := A w x 100
Method The water activity is determined by placing the

sample in a small airtight cup inside which the equilibrium
between the water in the solid and the headspace can be
established. The volume of the headspace must be small in
relation to the sample volume in order not to change the
sorption state of sample during the test. The equilibration as
a thermodynamic process takes time but may be accelerated
by forced circulation within the cell. The acquired water
activity value is only valid for the simultaneously determined
temperature. This requires a precise temperature-measuring
device as part of the equipment. Furthermore, the probe
must be thermally insulated to guarantee a constant
temperature during the test. The sensor measuring the
humidity of the headspace air aboye the sample is a key
component. Theoretically, all types of hygrometers can be
used, but for analytical purposes miniaturisation and
robustness are a precondition. The A w measurement may be
conducted using the dew pointlchilled mirror method 1 .
A polished, chilled mirror is used as a condensing surface.
The cooling system is electronically linked to a photoelectric
cell into which light is refiected from the condensing mirror.
An air stream, in equilibrium with the test sample, is
directed at the mirror, which cools until condensation occurs
on the mirror. The temperature at which this condensation
1 AOAC Intemational Official M ethod 978. 18.

V-A304 Appendix X 2014
D. Hydroxyl Value
Appendix X (Ph. Eur. method 2.5.3)
The hydroxyl value lOH is the number that express es in
A. Acetyl Value milligrams the quantity of potassium hydroxide required to
(No Ph. Eur. method) neutralise the acid combined by acylation in 1 g of the
The acetyl value of a substance is the number of mg of substance.
potassium hydroxide required to neutralise the acetic acid
liberated by the hydrolysis of 1 g of the acetylated substance. METHODA
Determine the saponification value, Appendix X G. Introduce the quantity of the substance to be examined
shown in T able 2.5.3 .-1 (m g) into a 150 mL acetylation
Acetylate by the following method. To 10 g in a 200-mL
flask fitted with an air condenser, unless another quantity is
Kjeldahl flask add 20 mL of acetie anhydride. Support the
prescribed in the monograph. Add the quantity of aeetie
flask on a sheet of heat resistant material in which a hole
anhydride solution R1 stated in Table 2.5 .3.-1 and anach the
about 4 cm in diameter has been cut and heat with a small,
air condenser.
naked flame, not more than 25 mm in height and which does
not impinge on the bottom of the flask. Boil gently under a
reflux air ·condenser for 2 hours, allow to cool, pour into Table 2.5 .3.-1
600 mL of water contained in a large beaker, add 0.2 g of Presumed value /OH Quantity of sample Volume of
(g) acetylating reagent (mL)
pumiee powder and boil for 30 minutes. Cool, transfer to a
separating funnel and discard the lower layer. Wash the 10 - 100 2.0 5.0
acetylated product with three or more 50-mL quantities of a 100 - 150 1.5 5.0
warm, saturated solution of sodium ehloride until the washings
150 - 200 1.0 5.0
are no longer acidic to litmus papel'. Finally shake with 20 mL
of warm water and remove the aqueous layer as completely as 200 - 250 0.75 5.0
possible. Pour the acetylated substance into a small dish, add 250·300 0.60 or 1.20 5.0 or 10.0
1 g of powdered anhydrous sodium sulfate, stir thoroughly and
300 - 350 1.0 10.0
filter through a dry, pleated filter paper. Determine the
saponification value of the acetylated substance. 350 - 700 0.75 15.0
Calculate the acetyl value from the expression 700 - 950 0.5 15.0
1335(b--a) /(1335 -a) where a is the saponification value of the
substance and b is the saponification value of the acetylated
substance. Heat the flask in a water-bath for 1 h keeping the level of the
water about 2.5 cm above the level of the liquid in the flask.
Withdraw the flask and allow to coo!. Add 5 mL of water R
through the upper end of the condenser. If a c10udiness
B. Acid Value appears add sufficient pyridine R to clear it, noting the
volume added. Shake the flask and replace in the water-bath
for 10 mino Withdraw the flask and allow to coo!. Rinse the
The acid value lA is the number that expresses, in milligrams
condenser and the walls of the flask with 5 mL of alcohol R,
the quantity of potassium hydroxide required to neutralise
previously neutralised to phenolphthalein solution R1 . Titrate
the free acids present in 1 g of the substance.
with 0.5 M alcoholie potassium hydroxide using 0.2 mL of
Dissolve 10.00 g of the substance to be examined, or the phenolphthalein solution RJ as indicator (nI mL of 0.5 M
quantity prescribed, (m g), in 50 mL of a mixture of equal alcoholie p otassium hydroxide). Carry out a blank test under
volumes of ethanol (96 per cent) R and light petroleum R3, the same conditions (n2 mL of 0.5 M aleoholic potassium
previously neutralised with 0.1 M potassium hydroxide or hydroxide) .
0.1 M sodium hydroxide, unless otherwise specified, using
0.5 mL of phenolphthalein solution R1 as indicator.
28.05(n2-nl) l
If necessary, heat to about 90 oC to dissolve the substance to l OH = m
+ A
be examined. When the substance to be examined has
dissolved, titrate with 0.1 M potassium hydroxide or 0.1 M
sodium hydroxide until the pink colour persists for at least 15 s
METHODB
(n mL of titrant). When heating has been applied to aid
dissolution, maintain the temperature at about 90 oC during Introduce the prescribed quantity of the substance to be
the titration. examined (m g) into a perfectly dry 5 mL conical flask fitted
with a ground-glass or suitable plastic stopper and add
lA = 5.610n 2.0 mL of propionie anhydride reagent R . Close the flask and
m shake gently to dissolve the substance. Allow to stand for 2 h
unless otherwise prescribed. Remove the stopper and transfer
the flask and its contents into a wide-mouthed 500 mL
C. Ester Value conical flask containing 25.0 mL of a 9 gIL solution of
aniline R in eyelohexane R and 30 mL of glacial acetie aeid R .
(Ph. E ur. method 2.5.2)
Swirl the contents of the flask, allow to stand for 5 min, add
The ester value lE is the number that expresses in milligrams 0.05 mL of erystal violet solution R and titrate with 0.1 M
the quantity of potassium hydroxide required to saponify the perehlorie acid until an emerald-green colour is obtained (ni
esters present in 1 g of the substance. It is calculated from mL of O. 1 M perehlorie acid). Carry out a blank test under the
the saponification value l s and the acid value lA: same conditions (f!2 mL of 0.1 M perehloric acid).
lE = 18 - lA
2014 Appendix X E V-A30S
Table 2.5.4.·2
Presumed Mass (g) Mass (g) lodine chloride
value 1, (corresponding (corresponding solution (mL)
to an excess oí to an excess oí
To take account of any water present, detennine this (y 150 per cent ICI) 100 per cent ICI)
per cent) by the semi-micro detennination of water (2.5.12). <3 10 lO 25
The hydroxyl value is then given by the equation: 3 8.4613 10.5760 25
¡OH = (hydroxyl value as detennined) - 31.1y
5 5.0770 6.3460 25
10 2.5384 3.1730 20
20 0.8461 1.5865 20
E. lodine Value 40 0.6346 0.7935 20
(Ph. Eur. method 2.5.4) 60 0.4321 0.5288 20
The iodine value JI is the number that expresses in grams the
80 0.3173 0.3966 20
quantity of halogen, caIculated as iodine, that can be fixed in
the prescribed conditions by 100 g of the substance. 100 0.2538 0.3173 20
When the monograph does not specify the method to be used, 120 0.2115 0.2644 20
method A is applied. Any change fram method A to method B is 140 0.1813 0.2266 20
validated.
160 0.1587 0.1983 20
METHODA 180 0.1410 0.1762 20
Unless otherwise prescribed, use the foIlowing quantities 200 0.1269 0.1586 20
(Table 2.5.4.-1) for the determination.
Table 2.5.4.-1 The mass of the sample is such that there wiII be an excess
Presumed value 1, Quantity oí sample (g)
of iodine chloride solution R of 50 per cent to 60 per cent of
the amount added, i.e. 100 per cent to 150 per cent of the
less than 20 1.0
amount absorbed.
20·60 0.5·0.25 Introduce the prescribed quantity of the substance to be
60· lOO 0.25·0.15 examined (m g) into a 250 mL flask fitted with a ground-
glass stopper and previously rinsed with glacial acetic acid R
more than 100 0.15·0.10
or dried, and dissolve it in 15 mL of a mixture of equal
volumes of cyclohexane R and glacial acetic acid R, unless
Introduce the prescribed quantity of the substance to be otherwise prescribed. If necessary, melt the substance before
examined (m g) into a 250 mL flask fitted with a ground- dissolution (melting point greater than 50 oC). Add very
glass stopper and previously dried or rinsed with glacial acetic slowly the volume of iodine chloride solution R stated in
acid R, and dissolve it in 15 mL of chlorofonn R unless Table 2.5.4.-2. Close the flask and keep it in the. dark for
otherwise prescribed. Add very slowly 25.0 mL of iodine 30 min, unless otherwise prescribed, shaking frequently.
bromide solution R. Close the flask and keep it in the dark for Add 10 mL of a 100 giL solution of potassium iodide R and
30 min unless otherwise prescribed, shaking frequently. 100 mL of water R. Titrate with O. 1 M sodium thiosulfate,
Add 10 mL of a 100 gIL solution of potassium iodide R and shaking vigorously until the yeIlow colour is almost
100 mL of water R. Titrate with 0.1 M sodium thiosulfate, discharged. Add 5 mL of starch solution R and continue the
shaking vigorously until the yeIlow colour is almost titration adding the 0.1 M sodium thiosulfate dropwise until
discharged. Add 5 mL of starch solution R and continue the the colour is discharged (n] mL of 0.1 M sodium thiosulfate).
titration adding the 0.1 M sodium thiosulfate dropwise until Carry out a blank test under the same conditions (n2 mL of
the colour is discharged (n¡ mL of 0.1 M sodium thiosulfate). 0. 1 M sodium thiosulfate).
Carry out a blank test under the same conditions (n2 mL of
0.1 M sodium thiosulfate). Jr = 1.269 (nz - nI)
m
J
r
= 1.269 (n2 - nI)
m Iodine MonochIoride Method
METHODB When the use of iodine flasks is prescribed, use flasks with a
Unless otherwise prescribed, use the foIlowing quantities nominal capacity of 250 mL and complying with British
(Table 2.5.4.-2) for the detennination. Standard 2735:1956 (Specification for iodine flasks), unless
otherwise specified.
Dissolve the specified quantity of the substance being
examined in 10 mL of dichloromethane in a dry iodine flask.
Add 20 mL of iodine monochloride solution, insert the stopper,
previously moistened with dilute potassium iodide solucion, and
aIlow to stand in the dark at 15° to 25° for 30 minutes. Place
15 mL of dilute potassium iodide solution in the top cup,
carefuIly remove the stopper, rinse the stopper and the sides
of the flask with 100 mL of water, shake and titrate with
O.IM sodium thiosulfate VS using starch mucilage, added
V-A306 Appendix X F 2014
towards the end of the titration, as indicator. At the same Titrate the solution with 0.01 M sodium chiosulface (VI mL),
time carry out the operation in exactly the same manner, but adding it gradually and with constant, vigorous shaking, until
without the substance being examined. the yellow iodine colour has almost disappeared. Add about
Calculate the iodine value from the expression 1.269 v/w 0.5 mL of scarch solution R1 and continue the titration, with
where v is the difference, in mL, between the titrations and constant shaking especially near the end-point, to liberate all
w is the weight, in g, of the substance taken. of the iodine from the solvent layer. Add the sodium
The approximate weight, in g, of the substance to be taken thiosulfate solution dropwise until the blue colour just
may be calculated by dividing 20 by the highest expected disappears.
iodine value. If more than half of the available halogen is D epending on the volume of 0.01 M sodiwn thiosulfate used,
absorbed, the test must be repeated, using a smaller quantity it may be necessary to titrate with 0.1 M sodium chiosulface.
of the substance. NOTE: there is a 15 sto 30 s delay in neutralising the starch
indicator for peroxide values of 70 and greater, due to the
tendency of trimethylpentane to f10at on the surface of the
aqueous medium and the time necessary to adequately mix
F. Peroxide Value the solvent and the aqueous titrant, thus liberating the last
Appendix X F traces of iodine. It is recommended to use 0.1 M sodium
thiosulfate for peroxide values greater than 150. A small
amount (0.5 per cent to 1.0 per cent (m/m)) of high HLB
The peroxide value I p is the number that expresses in emulsifier (for example polysorbate 60) may be added to the
milliequivalents of active oxygen the quantity of peroxide mixture to retard the phase separation and decrease the time
contained in 1000 g of the substance, as determined by the lag in the liberation of iodine.
methods described below.
Carry out a blank determination (Va mL). If the result of the
W'hen che monograph does noc specify che method to be used, blank determination exceeds 0.1 mL of titration reagent,
method A is applied. Any change from method A to method B is replace the impure reagents and repeat the determination.
validated.
METHODA Ip = 1000 (V1 - Va) c

Place 5.00 g of the substance to be examined (m g) in a m
250 mL conical flask fitted with a ground-glass stopper.
Add 30 mL of a mixture of 2 volumes of chloroform R and e concentration of the sodium thiosulfate solution in
3 volumes of glacial acetic acid R. Shake to dissolve the moles, per litre.
substance and add 0.5 mL of sacuraced pocassium iodide
solution R. Shake for exactly 1 min then add 30 mL of
waCer R. Titrate with O. 01 M sodium chiosulfate, adding the
titrant slowly with continuous vigorous shaking, until the G. Saponification Value
yellow colour is almost discharged. Add 5 mL of starch The saponification value is the number of mg of potassium
solution R and continue the titration, shaking vigorously, until hydroxide required to neutralise the free acids and to
the colour is discharged (ni mL of 0.01 M sodium chiosulface). saponify the esters in 1 g of the substance.
Carry out a blank test under the same conditions (n2 mL of
Use Method 1 unless otherwise specified in the monograph.
0.01 M sodium thiosulfate). The volume of 0.01 M sodium
thiosulface used in the blank titration must not exceed Method 1
0.1 mL.
Dissolve 35 to 40 g of potassium hydroxide in 20 ,mL of water
and add sufficient echanol (96%) to produce 1000 mL. Allow
to stand ovemight and pour off the c1ear liquido
METHODB Weigh 2 g of the substance into a 200-mLflask, add
Carry out che operations avoiding exposure to actinic light. 25.0 mL of the ethanolic solution of potassium hydroxide
Place 50 mL of a mixture of 2 volumes of crimechylpentane R and boil under a reflux condenser for 1 hour, rotating the
and 3 volumes of glacial acetic acid R in a conical f1ask and contents frequently . While the solution is still hot, titrate the
replace the stopper. Swirl the f1ask until the substance to be excess of alkali with O.5M hydrochloric acid VS using 1 mL of
examined (m g; see Table 2.5.5.-1) has dissolved. Using a phenolphthalein solution R1 as indicator. Repeat the operation
suitable volumetric pipette, add 0.5 mL of saturated potassium without the substance being examined.
iodide solution R and replace the stopper. Allow the solution Calculate the saponification value from the expression 28.05
to stand for 60 ± 1 s, thoroughly shaking the solution v/w where v is the difference, in mL, between the titrations
continuously, then add 30 mL of water R. and w is the weighr, in g, of substance taken.
Table 2.5.5.-1
Method 11
Expected peroxide Mass oC subslance
value 1, lo be examined (g) (Ph. Eur. method 2.5.6)
Olo 12 2.00 lo 5.00
The saponification value Is is the number that express es in
milligrams the quantity of potassium hydroxide required to
12 lO 20 1.20 lo 2.00 neutralise the free acids and to saponify the esters present in
20 lo 30 0.80 lo 1.20 1 g of the substance.
30 lo 50 0.500 lo 0.800 Unless otherwise prescribed, use the quantities indicated in
Table 2.5.6.-1 for the determination.
50 lO 90 0.300 lo 0.500
2014 Appendix X J V-A307
Table 2.5.6.-1 of freshly boiled ethanol (96%), previously neutralised to

Presumed vaIue Is Quantity of sample (g) phenolphthalein solution R 1. Titrate with 0.1 M ethanolic sodium
<3 20 hydroxide VS using phenolphthalein solution R1 as indicator.
3 to 10 12 to 15
If the volume of 0.1 M ethanolic sodium hydroxide VS required
does not exceed 0.1 mL, the amount of residue weighed is to
10 to 40 8 to 12 be taken as the unsaponifiable matter. Calculate the
40 to 60 5 to 8 unsaponifiable matter as a percentage of the substance being
60 to 100 3 to 5
examined.
If the volume of O.l M ethanolic sodium hydroxide VS required
100 to 200 2.5 to 3
exceeds 0.1 mL, the amount of residue weighed cannot be
200 to 300 1 to 2 taken as the unsaponifiable matter and the test must be
300 to 400 0.5 to 1 repeated.
Method II
Introduce the prescribed quantity of the substance to be (Ph. Eur. method 2.5.7)
examined (m g) into a 250 mL borosilicate glass f1ask fiued
The term "unsaponifiable matter" is applied to the
with a reflux condenser. Add 25.0 mL of 0.5 M alcoholic
substances non-volatile at 100-105 oC obtained by extraction
potassium hydroxide and a few glass beads. Attach the
with an organic solvent from the substance to be examined
condenser and heat under reflux for 30 min, unless otherwise
after it has been saponified. The result is calculated as
prescribed. Add 1 mL of phenolphthalein solution R1 and
per cent mlm.
titrate irnmediately (while still hot) with 0.5 M hydrochloric
acid (ni mL of 0.5 M hydrochloric acid). Carry out a blank Use ungreased ground-glass glassware.
test under the same conditions (nz mL of 0.5 M hydrochloric Introduce the prescribed quantity of the substance to be
acid). examined (m g) into a 250 mL f1ask fitted with a reflux
condenser. Add 50 mL of 2 M alcoholic potassium hydroxide R
I8 = 28.05 (n2 - n¡)
and heat on a water-bath for 1 h, swirling frequently. Cool to
m
a temperature below 25 oC and transfer the contents of the
f1ask to a separating funnel with the aid of 100 mL of
water R. Shake the liquid carefully with 3 quantities, each of
100 mL, of peroxide-free ether R. Combine the ether layers in
H. Unsaponifiable Matter another separating funnel containing 40 mL of water R, shake
The unsaponifiable matter is the percentage content, w/w, of gently for a few minutes, allow to separate and reject the
0 0
material not volatile at 100 to 105 that is obtained by aqueous phase . Wash the ether phase with 2 quantities, each
extraction with an organic solvent from the saponified of 40 mL, of water R then wash successively with 40 mL of a
substance being examined. 30 gIL solution of potassium hydroxide R and 40 mL of
Use Method I unless otherwise specijied in the monograph. water R; repeat this procedure 3 times. Wash the ether phase
Use ungreased ground-glass glassware for each method. several times, each with 40 mL of water R, until the aqueous
Method 1 phase is no longer alkaline to phenolphthalein. Transfer the
ether phase to a tared f1ask, washing the separating funnel
with peroxide-free ether R.
To 2.0 to 2.5 g of the substance being examined in a
250-mL f1ask add 25 mL of O.5M ethanolic potassium hydroxide Distil off the ether with suitable precautions and add 6 mL
and boil under a reflux condenser in a water-bath for 1 hour, of acetone R to the residue. Carefully remove the solvent in a
swirling the contents frequently. Wash the contents of the current of air. Dry to constant mas s at 100-105 ce. Allow to
f1ask into a separating funnel with the aid of 50 mL of water cool in a desiccator and weigh (a g).
and, while the liquid is still slightly warm, extract by shaking
. 100a
vigorously with three 50-rnL quantities of peroxide-free ether, Unsapomfiable matter =-m
- per cent
rinsing the f1ask with the first quantity of ether. Mix the ether
solutions in a separating funnel containing 20 mL of water.
Dissolve the residue in 20 mL of alcohol R, previously
(If the ether solutions contain solid suspended matter, filter
neutralised to phenolphthalein solution R and titrate with 0.1 M
them into the separating funnel through a fat-free filter paper
ethanolic sodium hydroxide. If the volume of 0.1 M ethanolic
and wash the filter paper with peroxide-free ether.) Gently
sodium hydroxide used is greater than 0.2 mL, the separation
rotate the separating funnel for a few minutes without violent
of the layers has been incomplete; the residue weighed
shaking, allow the liquids to separate and discard the
cannot be considered as "unsaponifiable matter". In case of
aqueous layer. Wash the ether solution by shaking vigorously
doubt, the test must be repeated.
with two 20-mL quantities of water and then treat with three
20-mL quantities of O.5M potassium hydroxide, shaking
vigorously on each occasion, each treatment being followed
by washing with 20 mL of water. Finally wash with successive
20-mL quantities of water until the aqueous layer is no longer
J. Determination of Cineole
alkaline to phenolphthalein solution R1 . Transfer the ether (Ph. Eur. method 2.8.11)
extract to a weighed f1ask, rinsing the separating funnel with Weigh 3.00 g of the oil, recently dried with anhydrous sodium
peroxide-free ether, distil the ether and add 3 mL of acetone to sulfate R, into a dry test-tube and add 2.10 g of melted
the ftask. With the aid of a gentle current of air, remove the cresol R . Place the tube in the apparatus for the determination
solvent completely from the ftask, which is almost entirely of freezing point (2.2.18) and allow to cool, stirring
immersed in boiling water and preferably held obliquely and continuously. When crystallisation takes place there is a small
rotated. Dry to constant weight at a temperature not rise in temperature . Note the highest temperature reached
exceeding 80 0 and dissolve the contents of the f1ask in 10 mL (ti) .
V-A308 Appendix X K 2014
Remelt the mixture on a water-bath at a temperature that and cenrrifuge. Transfer 30.0 mL of the clear supernatant
does not exceed tI by more than 5 oC and place the tube in liquid to a glass-stoppered 125 mL conical fiask. Add 1 mL
the apparatus, maintained at a temperature 5 oC below t1' of glacial acetic acid R and 0.5 g 10 1.0 g of potassium iodide R.
When crystallisation takes place, or when the temperature of Insert the stopper, swirl, and allow 10 stand for 25 min to
the mixture has fallen 3 oC below tI, stir continuously. Note 30 min in the dark. Add 1 mL of starch solution R and titrate
the highest temperature at which the mixture crystallises (t2)' with 0.002 M sodium thiosulfate until the starch-iodine colour
Repeat the operation until 2 highest values obtained for t2 do disappears. Carry out a blank determination. Not more than
not differ by more than 0.2 oc. If supercooling occurs, 1.4 mL of 0.002 M sodium thiosulfate is required
induce crystallisation by adding a small crystal of the (0.002 per cent, calculated as H 2 0 2 ).
complex consisting of 3.00 g of cineole R and 2.10 g of melted 1 mL of 0.002 M sodium thiosulfate is equivalent to 34 ¡lg of
cresol R. If t2 is below 27.4 oC, repeat the determination after oxidising substances, calculated as hydrogen peroxide.
the addition of 5.10 g of the complex.
The content of cineole corresponding to the highest
temperature observed ((2) is given in Table 2.8.11.-1.
If 5.10 g of the complex has been added, calculate the M. Essential Oils
cineole content per cent m/m from the expression:
2 (A - 50) Fatty Oils and Resinified Essential Oils in Essential
Oils
where A is the value found in Table 2.8.11.-1.
The content of cineole, corresponding 10 the highest
Allow 1 drop of the essential oil to fall onto filter papero
temperature observed (t2), is obtained, where necessary, by
The drop evaporates completely wíthin 24 h without leaving
interpolation.
any translucent or greasy spot.
Table 2.8.11.·1
Foreign Esters in Essential Oils
t, cineole per (, cineoleper (, cineole per (, cineole per (Ph. Eur. method 2.8.6)
oc centm/m oc centm/m oc cent m/m oc centm/m
Heat 1 mL of the essential oil for 2 min on a water-bath with
24 45.5 32 56.0 40 67.0 48 82,0
3.0 mL of a freshly prepared 100 gIL solution of potassium
25 47.0 33 57.0 41 68.5 49 84,0 hydroxide R in alcohol R. No crystals are formed within
26 48.5 34 58.5 42 70,0 50 86.0 30 min, even after cooling.
27 49.5 35 60,0 43 72,5 51 88,5
Odour and Taste of Essential Oils
28 50,5 36 61.0 44 74,0 52 91.0 (Ph. Eur. method 2.8.8)
29 52,0 37 62.5 45 76.0 53 93,5 Mix 3 drops of the essential oil with 5 mL of 90 per cent
30 53,5 38 63.5 46 78,0 54 96,0
V/V alcohol R and stir in 10 g of powdered sucrose R . The
odour and taste are similar to that of the plant or parts of the
31 54.5 39 65,0 47 80.0 55 99,0
plant from which the essential oil has been obtained.
Residue on Evaporation of Essential Oils

The residue on evaporation of an essential oil is the
K. Determination of Aldehydes percentage by mass of the oil which remains after evaporation
(No Ph. Eur. method) on a water-bath under the conditions specified below.
To 1 g of the oil in a glass-stoppered tube (approximately Apparatus The apparatus (see Figure 2.8.9.-1) consists of:
150 mm x 25 mm) add 5 mL of toluene and 15 mL of
- Water-bath with a cover having holes of 70 mm diameter;
alcoholic hydroxylamine solution, shake vigorously and titrate
immediately with O.5M potassium hydroxide in ethanol (60%) - Evaporating dish of heat-resistant glass which is inert to
VS until the red colour changes to yellow. Continue shaking the contents;
and neutralising until the full yellow colour of the indicator is - Desiccator.
permanent in the lower layer after shaking vigorously for
2 minutes and allowing to separa te; the reaction is complete 86
in about 15 minutes. This procedure gives an approximate
value for the aldehyde content of the oil.
Repeat this procedure, using as the colour standard for the
end point of the titration the titrated liquid of the first
determination with the addition of 0.5 mL of 0.5M potassium
hydroxide in ethanol (60%) VS. Calculate the content of
aldehydes from the second determination, using the
o
equivalent given in the monograph. LO
70
L. Oxidising Substances
Transfer 4.0 g to a glass-stoppered, 125 mL conical fiask and
Figure 2.8.9.-1.
add 50.0 mL of water R. Insert the stopper and swirl for
5 mino Transfer to a glass-stoppered 50 mL cenrrifuge tube Dimensions in millimetres
2014 Appendix X N V-A309
Method Weigh the evaporating dish after having heated it hydroxide. Add 10 mL of the oil to be examined, shake and
on the water-bath for 1 h and cooled it in the desiccator. allow to stand. Not more than 0.1 mL of 0.01 M hydrochloric
Weigh into the evaporating dish 5.00 g of the essential oil, acid is required to change the colour of the upper layer 10
unless otherwise prescribed. Heat the oil on the vigorously yellow.
boiling water-bath in a draught-free atmosphere for the
prescribed time. Allow to cool in the desiccator and weigh. Identification of Fixed Oils by Thin-Iayer
During the test, the level of water in the bath is maintained
Chromatography
about 50 mm beneath the level of the cover. (Ph. Eur. method 2.3.2)
METHODA
Solubility in Alcohol of Essential Oils
Thin-Iayer chromatography (2.2.27).
Test solution Unless otherwise prescribed, dissolve about
Place 1.0 mL of the essential oil in a 25 rnL or 30 mL glass-
20 mg (1 drop) of the fatty oi! in 3 mL of methylene
stoppered cylinder. Place in a constant temperature device,
chloride R.
maintained at a temperature of 20 ± 0.2 oc. Using a burette
of at least 20 mL capacity, add the alcohol of the strength Reference solution Dissolve about 20 mg (1 drop) of
prescribed in the monograph by increments of 0.1 mL until maize oil R in 3 mL of methylene chloride R.
solution is complete and then continue adding by increments Plate A suitable octadecylsilyl silica gel for high
of 0.5 mL to a total of 20 mL, shaking frequently and performance thin-Iayer chromatography as the coating
vigorously. Record the volume of alcohol added when a clear substance.
solution has been obtained and, if the solution becomes Mobile phase:
cloudy or opalescent before 20 mL of alcohol has been - mobile phase A : ether R;
added, record the volume added when the cloudiness or
- mobile phase B: methylene chloride R, glacial acetic acid R,
opalescence appears and, where applicable, the volume added
acetone R (20:40:50 VIV/V).
when the cloudiness or opalescence disappears.
Application 1 1lL.
If a clear solution has not been obtained when 20 mL of
alcohol of the prescribed strength has been added, repeat the Development Twice over a path of 0.5 cm with mobile
test using the next highest concentration of alcohol. phase A, then twice over a path of 8 cm with mobile
phase B.
An essential oil is said to be " soluble in n volumes and more
of alcohol of given strength t" when the clear solution in n Drying In airo
volumes remains clear when compared with the undi!uted oil Detection Spray with a 100 gIL solution of phosphomolybdic
after further addition of alcohol of the same strength up to a acid R in ethanol (96 per cent) R. Heat the plate at 120 oC
total of 20 volumes of alcohol. for about. 3 min and examine in daylight.
An essential oil is said to be "solúble in n volumes of alcohol The chromatogram obtained typically shows spots
of given strength t, becoming cloudy when diluted" when the comparable to those in Figure 2.3.2.-L
clear solution in n volumes becomes cloudy in nI volumes METHOD B
(nI less than 20) and stays so after further gradual addition Thin-layer chromatography (2.2.27).
of alcohol of the same strength up to a total of 20 volumes of
Test solution Unless otherwise prescribed, dissolve about
alcohol.
20 mg (1 drop) of the fatty oil in 3 mL of methylene
An essential oi! is said to be "soluble in n volumes of alcohol chloride R.
of given strength t with cloudiness between nI and n2
Reference solution Dissolve about 20 mg (1 drop) of
volumes" when the clear solution in n volumes becomes
maize oil R in 3 mL of methylene chloride R.
cloudy in nI volumes (nI less than 20) and stays so after
further gradual addition of alcohol of the same strengrh up to Plate A suitable octadecylsi!yl silica gel for high
a total of n2 volumes of alcohol and then becomes clear (n2 performance thin-Iayer chromatography as the coating
les s than 20). substance.
An essential oil is said to be "soluble with opalescence" Mobile phase methylene chloride R, glacial acetic acid R,
when the alcoholic solution shows a bluish tinge, similar to acetone R (20:40:50 VIV/V).
that of a standard of opalescence freshly prepared as follows: Application 1 1lL' as bands of 8 mm. A suitable automated
mix 0.5 mL of si/ver nitrate solution R2 and 0.05 mL of nitric apparatus may be used.
acid R; add 50 mL of a 12 mg/L solution of sodium Development Over a path of 7 cm.
chloride R; mix and allow to stand protected from light for Drying In air.
5 mino
Detection Treat with a 100 gIL solution of phosphomolybdic
Water in Essential Oils acid R in ethanol (96 per cent) R. Heat the plate at 120 oC
for 3 min and examine in daylight.
Mix 10 drops of the essential oil with 1 mL of carbon The chromatogram obtained typically shows zones
disulfide R . The solution remains clear on standing comparable to those in Figure 2.3.2.-2.
Test for Foreign Oils by Thin-Iayer

Chromatography
N. Fixed Oils (Ph. Eur. method 2.4.21)
(Ph. Eur. method 2.4.19) Examine by thin-Iayer chroma1Ography (2.2.27) using
In a test-tube mix 10 mL of recently distilled acetone R and kieselguhr G R as the coating substance. Impregnate a plate
0.3 mL of water R and add 0.05 mL of a 0.4 giL solution of by placing it in a chroma1Ographic tank containing the
bromophenol blue R in alcohol R. Neutralise the solution if necessary quantity of a mixture of 10 volumes of liquid
necessary with 0.01 M hydrochloric acid or 0.01 M sodium paraffin R and 90 volumes of light petroleum R so that the
V -A31 o Appendix X N 2014
2 3 4 5 7 8 9 10 11 12 13 14 15
l . arachis oi! 4. rapeseed oil 7. Iinseed oil 10. a!mond oi! 13. evening primrose oil
2. sesame oil 5. soya-bean oil 8. olive oil 11. wheat-germ oi! 14. safflower oi! (type I)
3. maize oi! 6. rapeseed oi! (erucic acid-free) 9. sunflower oil 12. borage oi! 15. safflower oi! (type II)
Figure 2.3.2.-1. - Chromatograms for the identification of fatty oi/s (method A)
....-: \ ~
1 2 _1~ ll,,,,,,,,,,,. .,,,,,,,,

1. arachis oi! 5. soya-bean oil 9. sunflower oH 13. evening primrose oi!
2. sesame oH 6. rapeseed oH (erucic acid-free) 10. almond oil 14. safflower oi! (type I)
3. maize oi! 7. Iinseed oi! 11. wheat-germ oH 15. safflower oil (type 11)
4. rapeseed oH 8. olive oH 12. borage oi! 16. hydrogenated arachis oi!
Figure 2.3.2.-2. - Chromatograms for the identification of fatty oils (method B)
plate dips abour 5 mm beneath the surfaee of the liquido eaeh of 10 mL, of water R; diseard the washings, dry the
When the impregnation mixture has risen by at least 12 cm ether over anhydrous sodium sulfate R and filter. Evaporate the
from the lower edge of the plate, remove the plate and allow ether on a water-bath. Use the residue to prepare the test
the solvent ro evaporate for 5 mino Carry out the solution. T he fatty aeids may also be obtained from the soap
ehromatography in the same direetion as the impregnation. solution prepared during the determination of the
Preparation of the m ixture offatty acids Heat 2 g of unsaponifiable matter.
the oil with 30 mL of 0.5 M alcoholic pOlassium hydroxide Test solution Dissolve 40 mg of the mixture of fatty aeids
under a reftux eondenser for 45 mino Add 50 mL of water R , obtained from the substanee ro be examined in 4 mL of
allow ro eool, transfer to a separating funnel and extraet with chlorofonn R .
three quantities, eaeh of 50 mL, of ether R . Diseard the ether Reference solution Dissolve 40 mg of the mixture of fatty
extraets, aeidify the aqueous layer with hydrochloric acid R aeids obtained from a mixture of 19 volurnes of maize oil R
and extraet with three quantities, eaeh of 50 mL, of ether R. and 1 volume of rapeseed oil R in 4 mL of chlorofonn R.
Combine the ether extraets and wash with three quantities,
2014 Appendix X N V -A311
Apply to the plate 3 flL of each solution. Develop over a to 50.0 mL with the same solvent. Commercially available
path of 8 cm using a mixture of 10 volumes of water R and mixtures of fany acid methyl esters may also be used.
90 volumes of glacial aeetie acid R. Dry the plate at 110 oC Column:
for 10 mino AlIow to cool and, unless otherwise prescribed, - material: fused silica, glass or quartz;
place the plate in a chromatographic chamber, with a tightly
fitting lid, that has previously been saturated with iodine - size: I = 10-30 m, 0 = 0.2-0.8 mm;
vapour by plaeing iodine R in an evaporating dish at the - starionary phase: macrogol 20 000 R (film thickness
bottom of the chamber. After sorne time brown or yellowish- 0.1-0.5 flm) or another suitable stationary phase.
brown spots become visible. Remove the plate and allow to Carrier gas helium for chromatography R 01' hydrogen for
stand for a few minutes. When the brown background colour ehromarography R.
has disappeared, spray with stareh solurion R . Blue spots Flow rate 1.3 mUmin (for a column 0 = 0.32 mm).
appear which may become brown on drying and again
Split ratio 1: 100 or les s, according to the internal
become blue after spraying with water R. The chromatogram
diameter ofthe column used (1:50 when 0 = 0.32 mm).
obtained with the test solution always shows a spot with an
R p of about 0.5 (oleic acid) and a spot with an RF of about Temperature:
0.65 (linoleic acid) corresponding to the spots in the - column: in isothermal conditions, 160-200 oC, according
chromatogram obtained with the reference solution. With to the length and type of column used (200 oC for a
sorne oils a spot with an Rp of about 0.75 may be present column 30 m long and coated with a layer of maerogol
(linolenic acid). By comparison with the spot in the 20000 R); if a linear temperature programming is
chromatogram obtained with the reference solution, verify necessary, raise the temperature of the column at arate of
the absence in the chromatogram obtained with the test 3 °C/min from 170 oC to 230 oC, for example;
solution of a spot with an RF of about 0.25 (erucic acid). - injeetion port: 250 oC;
- detector: 250 oc.
Test for Foreign Oils by Gas Chromatography
lnjeetion 1).lL.
The test for foreign oils is carried out on the methyl esters of
the fatty acids contained in the oil to be examined by gas System suitability when using the mixture of calibrating
chromatography (2.2.28). substances in Table 2.4.22.-1 or TabJe 2.4.22.-3:
METHODA
- resolution: minimum 1.8 between the peaks due to methyl
oleate and methyl stearate in the chromatogram obtained
This method is not applieable ro oils that contain glycerides of fatty
with reference solution (a);
acids with an epoxy-, hydroepoxy-, hydroperoxy-, cyclopropyl or
cyclopropenyl group, or those that eontain a large proportion of - signal-ro-noise ratio: minimum ~ for the peak due to methyl
fatty acids of ehain length less than 8 earbon aroms or ro oils with myristate in the chromatogram obtained with reference
an acid value greater than 2. O. solution (b);
Test solution When prescribed in the monograph, dry the - number of theorerical piares: minimum 30 000, calculated
oil to be examined before the methylation step. Weigh 1.0 g for the peak due to methyl stearate in the chromatogram
of the oil into a 25 mL round-bottomed flask with a ground- obtained with reference solution (a).
glass neck fitted with a reflux condenser and a gas port into Sysrem suitability when using the mixture of calibrating
the flask. Add 10 mL of anhydrous methanol R and 0.2 mL substances in Table 2.4.22.-2:
of a 60 giL solution of potassium hydroxide R in methanol R. - resolurion: minimum 4.0 between the peaks due to methyl
Attach the re flux condenser, pass nitrogen R through the caprylate and methyl caprate in the chromatogram
mixture at arate of about 50 mUmin, shake and heat to obtained with reference solution (a);
boiling. When the solution is cJear (usually after about - signal-ro-noise ratio: minimum 5 for the peak due to methyl
10 min), continue heating for a further 5 mino Cool the flask caproate in the chromatogram obtained with reference
under running water and transfer the contents to a solution (b);
separating funnel. Rinse the flask with 5 mL of hepcane R
- number of theorerical piares: mínimum 15 000, calculated
and transfer the rinsings to the separating funnel and shake.
for the peak due to methyl caprate in the chromatogram
Add 10 mL of a 200 gIL solution of sodiwn ehloride R and
obtained with reference solution (a).
shake vigorously. AlIow to separate and transfer the organic .
layer to a vial containing anhydrous sodium sulfate R. AlIow to Assessment of ehromatograms
stand, then filter. Avoid working conditions tending to give masked peaks
Referenee solution (a) Prepare 0.50 g of the mixture of (presence of constituents with smalJ differences between
calibrating substances with the composition described in one retention times, for example IÍnolenic acid and arachidic
of the 2.4.22 tables, as prescribed in the individual acid).
monograph (if the monograph does not mention a specific Qualitative analysis Identify the peaks in the
solution, use the composition described in T able 2.4.22.-1). chromatogram obtained with reference solution (c)
Dissolve in heptane R and dilute to 50.0 mL with the same (isothermal operating conditions or linear temperature
solvent. programming).
Referenee solution (b) Dilute 1.0 mL of reference When using isothermal operating conditions, the peaks may
solution (a) to 10.0 mL with heptane R . also be identified by drawing calibration curves using the
Referenee solution (e) Prepare 0.50 g of a mixture of chromatogram obtained with reference solution (a) and the
fatty acid methyl esters that corresponds in composition to information given in Tables 2.4.22 .-1, 2.4.22.-2 or 2.4.22.-3.
the mixture of fatty acids indicated in the monograph of the
substance to be examined. Dissolve in heptane R and dilute
V -A312 Appendix X N 2014
Table 2.4.22.'1. - Mixture of calibrating substances (for gas Identify the peaks in the chromatogram obtained with the
chromatography with capillary column and split inlet system, test solution by means of the straight line and the reduced
it is recommended that the component with the longest retention times. Equivalent chain lengths are given in
chain length of the mixture to be examined be added to the
calibration mixture, when the qualitative analysis is done Table 2.4.22.-4.
using calibration curves) Quantitative analysis In general, the normalisation
Mixture oí !he íollowing substances Composition (per cen! mlm) procedure is used in which the sum of the areas of the peaks
in the chromatogram, except that of the solvent, is set at
Methyllaurate R 5
100 per cent. The content of a constituent is calculated by
Methyl myristate R 5 determining the area of the corresponding peak as a
Methyl palmitate R 10 percentage of the sum of the areas of aH the peaks. Disregard
any peak with an area les s than 0.05 per cent of the total
Methyl stearate R 20
area.
Methyl arachidate R 40
In certain cases, for example in the presence of fatty acids
Methyl oleate R 20 with 12 or les s carbon atoms, correction factors can be
prescribed in the individual monograph to convert peak areas
in per cent mlm.
Table 2.4.22.-2. - Mixture of calibrating substances (for gas
chromatography with capillary column and split inlet system, Table 2.4.22.-4. - Equivalent chain lengths (Ihis value, which
it is recommended that the component with the longest is lo be calculated using calibration curves, is given as an
chain length ofthe mixture to be examined be added to the example for a column of macrogol20 000 R)
calibration mixture, when the qualitative analysis is done Fa!ty acid Equivalent chain length
using calibration curves)
Caproie acid 6.0
Mixture oí the following subslances Composition (per cent mlm)
Caprylic acid 8.0
Methyl caproate R 10
Capric acid 10.0
Methyl caprylate R 10
Laurie aeid 12.0
Methyl caprate R 20
Myristic acid 14.0
Methy//aurate R 20
Palmitie acid 16.0
Methyl myristate R 40
Palrnitoleic acid 16.3
Margarie acid 17.0
Table 2.4.22.-3. - Mixture of calibrating substances (for gas Stearic acid 18.0
chromatography with capillary column and split inlet system,
it is recommended that the component with the longest Oleie acid 18.3
chain length of the mixture to be examined be added to the Linoleic acid 18.8
calibration mixture, when the qualitative analysis is done
using calibration curves) Garnma·linolenic acid 19.0
Mixture of the íollowing substances Composition (per cent mlm) Alpha·linolenic acid 19.2
Methyl myristate R 5 Arachidic acid 20.0
Methyl pa/mitate R 10 Eicosenoie acid (¡¡ondoie acid) 20.2
Methyl stearate R 15 Arachidonic aeid 21.2

Methyl arachidate R 20 Behenic acid 22.0
Methyl o/eate R 20 Erucie acid 22.2
Methyl eicosenoate R 10 12-Oxostearic acid 22.7
Methyl behenate R 10 Ricinoleic acid 23.9
Methy/lignocerate R 10 12-Hydroxystearic acid 23.9

Li¡¡noeeric acid 24.0
Measure the reduced retention time (tI RJ of each peak in the Nervonic acid 24.2
chromatogram obtained with reference solution (a). tI R is the
retention time measured from the solvent peak and not from
the time of injection. Plot the straight line: METHOD B
This methad is not applicable to oils that contain glycerides oi fatty
lag (t~) = f (equivalent chain length) acids with an epoxy-, hydroepoxy-, hydroperoxy-, cyclopropyl or
cyclopropenyl group or to oils with an acid value greater than 2. O.
The logarithms of ti R of unsaturated acids are situated on Test solution Introduce 0.100 g of the substance to be
this line at points corresponding to non-integer values of examined into a 10 mL centrifuge tube with a screw cap.
carbon atoms known as 'equivalent chain lengths'; Dissolve with 1 mL of heptane R and 1 mL of dimethyl
the equivalent chain length is the length of the theoretical carbonate R and mix vigorously under gentle heating
saturated chain that would have the same tIRas the fatty acid (50-60 oC). Add, while stiH warm, 1 mL of a 12 gIL
to be identified. For example, linoleic acid has the same tI R solution of sodium R in anhydrous methanol R, prepared with
as the theoretical saturated fatty acid having 18.8 carbon the necessary precautions, and mix vigorously for about
atoms. 5 mino Add 3 mL of distilled water R and mix vigorously for
2014 Appendix X P V-A313
about 30 s. Centrifuge for 15 min at 1500 g. Inject 1 ¡.tL of Test solution (b) To 5.0 mL of test solution (a) add
the organic phase . 1.0 mL of a 2.5 gIL solution of p-anisidine R in glacial aeetic
Reference solutions and assessrnent of chromatograms acid R, shake and store protected from light.
Where there is no specific prescription in the individual Reference solution To 5.0 mL of trimethylpentane R add
monograph, proceed as described under Method A. 1.0 mL of a 2.5 gIL solution of p-anisidine R in glacial aeetic
Column: acid R, shake and store protected from light.
- material: fused silica; Measure the absorbance (2.2.25) of test solution (a) at the
maximum at 350 nm using trimethylpentane R as the
- size: 1 = 30 m, 0 = 0.25 mm;
compensation liquido Measure the absorbance of test solution
- stationary phase: macrogol 20 000 R (film thickness
(b) at 350 nm exactly 10 min after its preparation, using the
0.25 ¡.tm). reference solution as the compensation liquido
Carrier gas helium for chromalOgraphy R. Calculate the anisidine value from the expression:
Flow rate 0.9 mUmin.
Split ratio 1:100. 25 X (1.2A¡ - A 2 )
Temperature m
absorbance of test solution (b) at 350 nm,

Time Temperature
absorbance of test solution (a) at 350 nm,
(min) (oC)
mass of the substance to be examined in test solution
Column 0-15 100
(a), in grams.
15 - 36 100 ~ 225
36 - 61 225
Injection port 250

P. Oils Rich in Omega-3-acids
Detector 250
1. Composition of Fatty Acids
Detection Flame ionisation. The assay may be used for quantitative determination of the EPA
Injection 1 ¡.tL. and DHA content in omega-3-containing products of fish oil in
METHOD e different coneentrations. The method is applicable to tnglyeerides or
This method is not applicable lO oils that contain glycerides of fatty ethyl esters and the results are expressed as tnglycerides or ethyl
acids with epoxy-, hydroepoxy-, hydroperoxy-, aldehyde, kelOne, esters, respective/y.
cyclopropyl and cyclopropenyl groups, and conjugated
polyunsaturated and acetylenic compounds because of partíal or
EPA andDHA
complete destruction of these groups. Gas chromatography (2.2.28). Cany out the operations as
Test solution Dissolve 0.10 g of the substance to be rapidly as possible, avoiding exposure lO actinic light, oxidising
examined in 2 mL of a 20 gIL solution of sodium hydroxide R agents, oxidation eatalysts (for example, eopper and iron) and airo
in methanol R in a 25 mL conical flask and boj] under a The assay is carried out on the methyl or ethyl esters of (all-
reflux condenser for 30 mino Add 2.0 mL of boro n trifluoride- Z)-eicosa-5,S,1l,14,17-pentaenoic acid (EPA; 20:5 n-3) and
methanol solution R through the condenser and boj] for (all-Z)-docosa-4, 7,10,13,16, 19-hexaenoic acid (DHA; 22:6
30 mino Add 4 mL of heptane R through the condenser and n-3) in the substance to be examined.
boil for 5 mino Cool and add 10.0 mL of saturated sodium Internal standard Methyl tricosa/wate R.
chloride solution R, shake for about 15 s and add a quantity of Test solution (a)
saturated sodium ehloride solution R such that the upper phase
A. Dissolve the mass of sample to be examined according
is brought into the neck of the flask. Collect 2 mL of the
to Table 2.4.29 .-1 and about 70.0 mg of the internar
upper phase, wash with 3 quantities, each of 2 mL, of
standard in a 50 mgIL solution of butylhydroxylOluene R
water R and dry over anhydrous sodium sulfate R .
in trimethylpentane R and dilute to 10.0 mL with the
Reference solutions, chromatographic procedure and same solution. Gentle heating (up to 60 oC) may be
assessrnent of chromatograms Where there is no applied to dissolve the internal standard.
specific prescription in the individual monograph, proceed as
described under Method A. TabIe 2.4.29.-1.
Approximate sum EPA + DHA Mass of sample to be examined
(per cent) (g)
30·50 0.4·0.5
O. Anisidine Value
50·70 0.3
The anisidine value is defined as 100 times the optical 70·90 0.25
density measured in a 1 cm cell of a solution containing 1 g
of the substance to be examined in 100 mL of a mixture of Ethyl esters are now ready for analysis. For triglycerides
solvents and reagents according to the following method. continue as described in step B.
Carry out the operations as rapidly as possible, avoiding exposure B. Introduce 2.0 mL of the solution obtained in step A ¡nto
to actinie light. a quartz tube and evaporate the solvent with a gentle
Test solution (a) Dissolve 0.500 g of the substance to be current of nitrogen R. Add 1.5 mL of a 20 gIL solution
examined in trimethylpentane R and dilute to 25.0 mL with of sodium hydroxide R in methanol R, cover with
the same solvento nitrogen R, cap tightly with a polytetrafluoroethylene-
V-A314 Appendix X P 2014
lined cap, mix and heat on a water-bath for 7 mino Temperature:

Allow to coo!. Add 2 mL of boron trichloride-methanol
solution R, cover with nitrogen R, cap tightly, mix and Time Temperature
(min) (oC)
heat on a water-bath for 30 mino Cool ro 40-50 oC, add
1 mL of trimethylpentane R, cap and shake vigorously for Column 0-2 170
at least 30 S. Immediately add 5 mL of a saturated 2 - 25.7 170 -> 240

sodium chloride solution R, cover with nitrogen R, cap and 25.7·28 240
shake thoroughly for at least 15 S. Transfer the upper
layer to a separate tube. Shake the methanol layer once Injection por! 250
more with 1 mL of trimethylpentane R. Wash the Detector 270
combined trimethylpentane extracts with 2 quantities,
each of 1 mL, of water R and dry over anhydrous sodium Deteetion Flame ionisation.
sulfate R. Prepare 3 solutions for each sample.
Injeetion 1 I1L, twice.
Test solution (b) Dissolve 0.300 g of the sample ro be
System suitability:
examined in a 50 mgIL solution of butylhydroxytoluene R in
trimethylpentane R and dilute ro 10.0 mL with the same - in the chromatogram obtained with reference solution (b),
solution. Proceed as described for test solution (a) . the area per cent composition increases in the following
order: methyl palmitate, methyl stearate, methyl
Referenee solution (aJ Dissolve about 70.0 mg of the
arachidate, methyl behenate; the difference between the
internal standard and 90.0 mg of eicosapentaenoic acid ethyl
percentage area of methyl palmitate and that of methyl
ester CRS in a 50 mgIL solution of butylhydroxytoluene R in
behenate is les s than 2.0 area per cent units;
trimethylpentane R and dilute ro 10.0 mL with the same
solution. Gentle heating (up ro 60 oC) may be applied to - resolution: minimum of 1.2 between the peaks due ro
dissolve the internal standard. docosahexaenoic acid methyl ester and to tetracos-
15-enoic acid methyl ester in the chromatogram obtained
Referenee solution (a:J Dissolve 60.0 mg of
with reference solution (e);
docosahexaenoic acid ethyl ester CRS and about 70.0 mg of the
internal standard in a 50 mgIL solution of - in the chromatogram obtained with test solution (a), the
butylhydroxytoluene R in trimethylpentane R and dilute to peaks due ro methyl tricosanoate and any
10.0 mL with the same solution. Gentle heating (up ro heneicosapentaenoic acid methyl ester or ethyl ester
60 oC) may be applied to dissolve the internal standard. (C21:5) present when compared with the chromatogram
obtained with test solution (b) are clearly separated (if
For both reference solution (al) and reference solution (a2)
not, a correction factor has to be used).
proceed as described for test solution (a) step A when
analysing ethyl esters. For analysis of triglycerides, continue Calculate the percentage content of EPA and DHA using the
with step B in the same manner as for test solution (a) and following expression and taking into account the assigned
prepare 3 solutions for each sample. value of the reference substances:
Referenee solution (b) Into a 10 mL volumetric fiask A x,3 mI mx,r 1
dissolve 0.3 g of methyl arachidate R, 0.3 g of methyl Ax x - - x - x -- x - x ex 100
m x ,3 Al Ax ,r m2
behenate R, 0.3 g of methyl palmitate R and 0.3 g of methyl
stearate R in a 50 mgIL solution of butylhydroxytoluene R in
trimethylpentane R and dilute to 10.0 mL with the same mI mass of the internal standard in test solution (a), in
solution. milligrams;
m2 mass of the sample to be examined in test solution
Referenee solution (e) Into a 10 mL volumetric fiask
(a), in milligrams;
dissolve a sample containing about 55.0 mg of
mx ,3 mass of the internal standard in reference solution
docosahexaenoic acid methyl ester R and about 5.0 mg of
(al) (EPA determination), or in reference solution
tetracos-15-enoic acid methyl ester R in a 50 mglL solution of
(a2) (DRA determination), in milligrams;
butylhydroxy toluene R in trimethylpentane R and dilute to
mx,r mass of eicosapentaenolic acid ethyl ester CRS in
10.0 mL with the same solution.
reference solution (al) or docosahexaenoic acid ethyl
Column: ester CRS in reference solution (a2), in milligrams;
- material: fused silica; Ax area of the peak due to eicosapentaenoic acid ester
- dimensions: 1 = at least 25 m, 0 = 0.25 mm; or docosahexaenoic acid ester in the chromatogram
- stationary phase: bonded macrogol 20 000 R (film thickness obtained with test solution (a);
0.2 ¡.tm) . A x,r area of the peak due to eicosapentaenoic acid ester
in the chromatogram obtained with reference
Carrier gas hydrogen ¡or chromatography R or helium ¡or
solution (al) or to docosahexaenoic acid ester in the
chromatography R.
chromatogram obtained with reference solution (a2);
Flow rate 1 mUmin. Al area of the peak due ro the internal standard in the
Split ratio 1:200, alternatively splitless with temperature chromatogram obtained with test solution (a);
control (sample solutions need to be diluted 1/200 with a A x,3 area of the peak due to the internal standard in the
50 mglL solution of butylhydroxytoluene R in chromatogram obtained with reference solution (al)
trimethylpentane R before injection). (EPA determination) or with reference solution (a2)
(DRA determination);
C conversion factor between ethyl ester and
triglycerides,
e = 1.00 for ethyl esters,
e = 0.954 for EPA,
e = 0.957 for DHA.
2014 Appendix X P V-A315
Total Omega-3-Acids Evaporate to dryness under a gentle stream of nitrogen with

From the assay for EPA and DHA, calculate the percentage careful heating. Dissolve the residue in 600 ¡.¡L of ethyl
content of the total omega-3 acids using the following acetate R.
expression and identifying the peaks from the Depending on the expected cholesterol content in the oil, the
chromatograms: solution is further diluted as follows:
- content less than 3 mg!g: dilute 200 ¡.¡L of the solution to
EPA+DHA + An-3 (EPA+DHA)
2.0 mL with ethyl acerare Rj
AEPA +ADHA
- content greater than or equal to 3 mg!g: dilute 20 ~IL of
EPA percentage content of EPA; the solution to 2.0 mL with ethyl acerare R.
DHA percentage content of DHA; Reference solution (a) Transfer 1.0 mL of the internal
A n- 3 sum ofthe areas ofthe peaks due to C18:3 n-3, standard stock/working solution and 1.0 mL of the
C18:4 n-3, C20:4 n-3, C21:5 n-3 and C22:5 n-3 cholesterol stock/working solution, depending on the
esters in the chromatogram obtained with test expected cholesterol content in the oil (see Table 2.4 .32.-1),
solution (b); to a 15 mL quartz tube and continue as described for the
area of the peak due to EPA ester in the test solution, starting with "Evaporate to dryness on a
chromatogram obtained with test solution (b); heating block ... ".
area of the peak due to DHA ester in the Reference solution (b) Mix 1.0 mL of the internal
chromatogram obtained with test solution (b). standard stock solution, 1.0 mL of the cholesterol stock
solution and 2.0 mL of the a-tocopherol solution in a
2. Total Cholesterol in Oils Rich in Omega-3-Acids suitable fiask. Evaporate to dryness under a gentle stream of
(Ph. Eur. method 2.4.32) nitro gen with careful heating. Dissolve the residue in ethyl
This method may be used for the quantitative determination of the acetate R and dilute to 50.0 mL with the same solvent.
sum of free and esterified cholesterol in products of fish oils rich in Dilute 1.0 mL ofthis solution to 10.0 mL with ethyl
omega-3 acids (as ethyl esters or triglycerides). acetate R. The solution may be stored in a deep-freezer for
up to 6 months.
Gas chromatography (2.2.28).
Internal standard stock solution Dissolve 0.15 g of Table 2.4.32.-1. - Preparation of the test and reference
(5rx)-cholestane R in heptane R and dilute to 50.0 mL with solutions
the same solvento
Reference Reference
Internal standard working solution Prepare the solution Test solution
solution (a) solution (b)
immediately before use. Dilute 1.0 mL of the internal standard less greater less greater
stock solution to 10.0 mL with heptane R. than than or than than or
3 rngjg equal to 3 rngjg equal to
Cholesterol stock solution Dissolve 30.0 mg of 3 rngjg 3rngjg
cholesterol R in heptane R and dilute to 10.0 mL with the Internal
same solvent. The solution is dispensed into gas standard stock + + +
chromatography vials and may be stored in a deep-freezer for solution
up to 6 months. Internal
standard + +
Cholesterol working solution Prepare the solution working
immediately before use. Dilute 1.0 mL of the cholesterol stock solution
solution to 10.0 mL with heptane R. Cholesterol + +
stock solution
a-Tocopherol solution Dilute 15.0 mg of
Cholesterol
rx-tocopherol CRS to 10.0 mL with heptane R. working +
Test solution Weigh 0.100 g of the substance to be solution
examined into a 15 mL quartz tube. Add 1.0 mL of the a-Tocopherol
+
internal standard stock/working solution, depending on the solution
expected cholesterol content in the oil (se e Table 2.4.32.-1).
Evaporate to dryness on a heating block at 50 oC under a Column:
gentle stream of nitro gen, while mixing. Add 0.5 mL of a - size: l = 30 m, 0 = 0.25 mm (film thickness 0.25 ¡.¡m);
50 per cent solution of potassium hydroxide R and 3.0 mL of - stationary phase: poly(dimethyl) (diphenyl)siloxane R.
ethanol (96 per cene) R. Fill the tube with nitrogen R and cap .
Further heat on the heating block at 100 oC for 1 h with
stirring. Cool for about 10 min and add 6 mL of distilled Flow rate 1.3 mUmin.
water R. Extract with 4 quantities, each of 2.5 mL, of ether R, Temperature:
mixing each time for 1 min using a vortex mixer. Transfer
the ether phase to a large centrifuge tube or a separating Time Temperature
funnel and wash the combined extracts with 5 mL of distilled (min) ('C)
water R, mixing carefully a fixed number of times, Colurnn o· 1 170
e.g. 60 times . Discard the aqueous phase, add 5 mL of a 1· 38 170 -> 320
3 per cen! solution of potassium hydroxide R to the ether
phase and mix carefully 20 times. Discard the aqueous 38·40 320
phase, add another 5 mL of distilled water R and mix carefully lnjection port 320
a further 20 times . Transfer the ether phase into a small Detector 300
centrifuge tube, avoiding any transfer of water. If an
emulsion forms during the process, add a small amount of
sodium chloride R to get a separation of the phases. Detection Flame ionisation.
V -A316 Appendix X Q 2014
Injeetion 1 1lL. quantities, each of 100 mL, of peroxide-free ether R. Combine

System suitability reference solution (b): the ether layers in another separating fu=el containing
40 mL of water R, shake gently for a few minutes, allow to
- resolution: minimum 1.2 between the peaks due to
separate and reject the aqueous phase. Wash the ether phase
cholesterol and IX-tocopherol.
with several quantities, each of 40 mL, of water R, until the
Calculate the content of total cholesterol, expressed as aqueous phase is no longer alkaline to phenolphthalein.
milligrams of cholesterol per gram of oil, using the following Transfer the ether phase to a tared fiask, washing the
expression: separating funnel with peroxide-free ether R . Distil off the
ether with suitable precautions and add 6 mL of aeelOne R to
the residue. Carefully remove the solvent in a current of
nitrogen R. Dry to constant mass at 100-105 oc. Allow to
cool in a desiccator and weigh. Transfer the residue to a
Al area of the peak due to cholesterol in the small test tube with methylene ehloride R. Evaporate under a
chromatogram obtained with the test solution; stream of nitrogen R to a volume of about 1 mL. Depending
A2 area of the peak due to (51X)-cholestane in the on the unsaponifiable content of the oil, adapt the final
chromatogram obtained with the test solution; concentration of the solution to 25-50 mglmL.
mI mas s of the substance to be examined in the test
Test solution (b) Treat 5.00 g of rapeseed oil R as
solution, in grams;
prescribed for the substance to be examined, begi=ing at
m2 mass of (51X)-cholestane in the internal standard stock
the words "Add 50 mL of 2 M alcoholie potassium
solution, in grams;
hydroxide R".
F 20 for oils with an expected cholesterol content
greater than or equal to 3 mglg; 2 for oils with an Test solution (e) Treat 5.00 g of sunflower oil R as
expected cholesterol content less than 3 mglg; prescribed for the substance to be examined, begi=ing at
R response factor. the words "Add 50 mL of 2 M aleoholie potassium
hydroxide R".
Calculate the response factor R using the following
Referenee solution Dissolve 25 mg of eholesterol R and
expression:
10 mg of betulin R in 1 mL of methylene ehloride R.
Use a separate plate for each test solution. Apply as a band
of 10 mm, at 20 mm from the base and 10 mm from the left
edge, 10 IlL of the reference solution and as bands of
area of the peak due to cholesterol in the 150 mm, at 20 mm from the base, 0.5 mL of test solutions
chromatogram obtained with reference solution (a); (a), (b) or (e). Develop over a path of 17 cm using a mixture
area of the peak due to (51X)-cholestane in the of 35 volumes of ether R and 65 volumes of hexane R.
chromatogram obtained with reference solution (a); Dry the plates in a current of nitrogen R. Spray the plates
mas s of cholesterol in the cholesterol stock solution, with a 2 gIL solution of diehlorofluoreseein R in anhydrous
in grams. ethanol R and examine in ultraviolet light at 254 nm.
The chromatogram obtained with the reference solution
shows bands due to cholesterol and betulin.
The chromatograms obtained with the test solutions show
Q. Sterols in Fatty Oils bands with similar RF values due to sterols. From each of the
chromatograms, remove an area of coating corresponding to
the area occupied by the sterol bands and additionally the
When the monograph does not speeify the method lO be used,
area of the zones 2-3 mm aboye and below the visible zones
method A is applied. Any ehange frommethod A lO method B
corresponding to the reference solution. Place separately in
must be validated.
three 50 mL fiasks. To each fiask add 15 mL of methylene
MethodA ehloride R and heat under refiux with stirring, for 15 mino
Filter each solution through a sintered-glass filter (40) (2.1.2)
Separation of the sterol fraction (TLC) or suitable filter paper and wash ea eh filter with 3 quantities,
Prepare the unsaponifiable matter and then isolate the sterol each of 15 mL, of methylene ehloride R. Place the combined
fraction of the fatty oil by thin-Iayer chromatography filtrate and washings from each filter separately in 3 fiasks,
(2.2.27), using a TLC siliea gel plate R with a 0.2 mm to evaporate under a stream of nitrogen R to 5-10 mL. Transfer
0.5 mm layer. to a small test tube and evaporate to dryness under a stream
Test solution (a) In a 150 mL fiask fitted with a refiux of nitrogen R.
condenser, place a volume of a 2 giL solution of betulin R in
methylene ehloride R containing betulin corresponding to Determination ofthe sterols (GC)
about 10 per cent of the sterol content of the sample used Gas chromatography (2.2.28). Carry out che operations
for the determination (e.g. in the case of olive oil add protected from humidity and prepare the solutions immediatery
500 IlL, in the case of other vegetable oils add 1500 IlL of before use.
the betulin solution). If the monograph requires the Test solution To the sterols separated from the substance
percentage content of the individual sterols in the sterol to be examined by thin-Iayer chromatography add a freshly
fraction, the addition of betulin may be omitted. Evaporate prepared mixture of 0.04 mL of ehlorotrimethylsilane R ,
to dryness under a current of nitrogen R. Add 5.00 g (m ) of 0.1 mL of hexamethyldisilaz ane R and 0.5 mL of anhydrous
the substance to be examined. Add 50 mL of 2 M alcoholie pyridine R . Allow to stand for at least 5 min and use the
potassiwn hydroxide R and heat on a water-bath for 1 h, liquid phase.
swirling frequently. Cool to a temperature below 25 oC and
Reference solution (a) To 9 parts of the sterols separated
transfer the contents of the fiask to a separating fu=el with
from rapeseed oil R by thin-Iayer chromatography add I pan
100 mL of water R. Shake the liquid carefully with 3
2014 Appendix X Q V-A317
of cholesterol R. To the mixture add a freshly prepared The peak due to the internal standard (betulin) must be
mixture of 0.04 mL of chlorotrimethylsi/ane R, 0.1 mL of c1early separated from the peaks due to the sterols to be
hexamethyldisilazane R and 0.5 mL of anhydrous pyridine R. determined.
Allow to stand for at least 5 min and use the liquid phase. For the chromatogram obtained with the test solution,
Reference solution (b) To the sterols separated from identify the peaks and calculate the percentage content of
sunfiower oil R by thin-Iayer chromatography add a freshly each sterol in the sterol fraction of the substance to be
prepared mixture of 0.04 mL of chlorotrimethylsilane R, examined using the following expression:
0.1 mL of hexamethyldisilazane R and 0.5 mL of anhydrous
pyrídine R. Allow to stand for at least 5 min and use the A
liquid phase.
S x 100
Column:
- material: fused silica; A area of the peak due to the component to be
determined;
- size: 1 = 20-30 m, 0 = 0.25-0.32 mm;
S sum of the areas of the peaks due to the components
- stationary phase: poly[methyl(95)phenyl(5)Jsiloxane R or indicated in Table 2.4.23.-1 ; disregard the peak due
poly(cyanopropyl) (7) (phenyl) (7) (methyl) (86) siloxane R (film to betulin.
thickness 0.25 ¡.tm).
If required in the monograph, calculate the content of each
Carrier gas hydrogen for chromatography R or helium for
sterol in milligrams per 100 grams of the substance to be
chromatography R.
examined using the following expression:
Linear velocity 30-50 cm/s (hydrogen) or 20-35 cm/s
(helium).
Split ratio 1 :50 (hydrogen) or 1: 100 (helium).
Temperature:
- column: 260 oC; A area of the peak due to the component to be
determined;
- injection port: 280 oC; Al area of the peak due to betulin;
- detector. 290 oc. m mass of the sample of the substance to be examined,
Detection Flame ionisation. in grams;
Injection 1 ¡.tL. mi mass of betulin R added, in milligrams.
Identification of peaks The chromatogram obtained with
reference solution (a) shows 4 principal peaks corresponding
MethodB
to cholesterol, brassicasterol, campesterol and B-sitosterol
and the chromatogram obtained with reference solution (b) Preparation of the unsaponifiable matter
shows 4 principal peaks corresponding to campesterol,
Prepare the unsaponifiable matter according to the method
stigmasterol, B-sitosterol and Ll7 -stigmastenol. The relative
stated in the test for unsaponifiable matter of the monograph
retentions of the sterols with reference to B-sitosterol (main
on the substance to be examined. Failing this, prepare the
peak) are given in Table 2.4.23.-1.
unsaponifiable matter according to the method described in
chapter 2.5.7. Unsaponifiable matter. After the final
Table 2.4.23.-1. - Relafive refentions of sterols with reference neutralisation step, evaporate the ethanol, then add 6 mL of
fa f3-sitosterol for 2 different columns acetone R and evaporate the solvento Dry the residue at
Poly(cyanopropyl)(7)- Poly[methyl(95)- 100-105 oC. It is not necessary to dry to constant mass.
(phenyl)( 7)- phenyl(5)lsiloxane
(methyl)(86)siloxane Simultaneously prepare under the same conditions the
Cholesterol 0.64 0.63 unsaponifiable matter of sunfiower oil R. This will in
particular serve to locate the sterol fraction to be collected .
Brassicasterol 0.70 0.71
24-Methylenecholesterol 0.79 0.80 Separation of the sterol fraction (Le)
Campes te rol 0.82 0.81 Liquid chromatography (2.2.29).
Campestanol 0.83 0.82 Test solution Take up the residue with 3 quantities, each
of 4 mL, of the solvent used during the preparation of the
Stigmasterol 0.87 0.87
unsaponifiable matter (generally ether R or light petroleum R)
/::'7·Campesterol 0.93 0.92 and transfer to a 15 mL tube. Evaporate to dryness under a
/::'5.23-Stigmastadienol 0.95 0.95 current of nitrogen R. Dissolve the residue in a volume of
mobile phase sufficient to obtain a solution with an
Clerosterol 0.96 0.96
approximate concentration of 40 mg/mL. Add a few drops of
~Sitosterol 2-propanol RI to improve the solubility (3 drops are normally
Sitostanol 1.01 1.02 sufficient to ensure complete solubilisation). Filter through a
membrane filter (nominal pore size 0.45 ~lm).
/::'5·Avenasterol 1.03 1.03
Reference solution Proceed as described for the test
/::'5,24-Stigmastadienol 1.09 1.08 solution with the unsaponifiable matter obtained with
Ll7-Stigmastenol w 1.13 1.12 sunfiower oil R.
/::'7-Avenasterol 1.18 1.16 Precolumn:
- size: 1 = 5 mm, 0 = 4.6 mm;
Betulin 1.4 1.4
- stationary phase: si/iea gel for ehromatography R (5 ¡.tm) with
O) This sterol may also be referred to as 117-stigmasterol in literature. a pore size of 6 nm.
V -A318 Appendix X Q 2014
Column: obtained with the referenee solution and the relative

- size: 1 = 0.25 m, 0 = 4.6 mm; retentions with referenee to ~-sitosterol (main peak) given in
Table 2.4 .23.-1.
- stationary phase: silica gel for chmmatography R (5 ¡.tm) with
a pore size of 6 nm. System suitability Referenee solution:
Mobile phase 2-propanol Rl, hexane R (1 :99 VN) . - resolution: minimum 4.0 between the peaks due to
eampesterol and stigmasterol.
Flow rate 1 mUmin.
Calculate the pereentage eontent of eaeh sterol in the sterol
Detection Speetrophotometer at 210 nm.
fraetion of the substanee to be examined using the following
Injection 50 [!L. expression:
Identification 01 the peaks due to sterols The sterol
fraetion e1utes at the end of the ehromatogram. Loeate the A
fraetion to be eolleeted using the ehromatogram obtained S x 100
with the referenee solution, which shows 2 principal peaks
eluting approximately between 23 min and 32 mino Colleet A area of the peak due to the eomponent to be
the fraetion at the detector outlet in a 15 mL tube with a determined;
serew cap. Evaporate the solvent under a eurrent of S sum of the areas of the peaks due to the eomponents
nitrogen R. indieated in Table 2.4 .23.-1, exeept betulin.
Deterrnination of the sterols (GC)

Gas ehromatography (2.2.28).
Test solution Dissolve the residue of the sterol fraetion
obtained with the test solution in the previous LC step in
0.2 mL of anhydrous pyridine R and 0.2 mL of a mixture of
1 volume of chlorotrimethylsilane R and 99 volumes of
N,O-bis(trimethylsilyl)trifiuoroacetamide R. Stopper the tube
tightly and heat at 80 oC for 20 mino Allow to eool and use
the liquid phase.
Relerence solution Dissolve the residue of the sterol
fraetion obtained with the referenee solution in the previous
LC step in 0.2 mL of anhydrous pyridine R and 0.2 mL of a
mixture of 1 volume of chlorotrimethylsilane R and
99 volumes of N,O-bis(trim ethylsilyl)trifiuoroacetamide R.
Stopper the tube tightly and heat at 80 oC for 20 mino Allow
to eool and use the liquid phase.
A standard of eholesterol (cholesterol R) may also be used,
alone or as a mixture with the sterol fraetion of sunflower oi!.
Proeeed with derivatisation as deseribed for the test solution.
Column:
- material: fused siliea;
- size: 1 = 30 m, 0 = 0.25 mm;
- stationary phase: poly{methyl(95)phenyl(5)Jsiloxane R (film
thiekness 0.25 ¡.tm).
Carrier gas helium for ehromatography R.
Split ratio 1:25.
Temperature:
Time Temperature
(min) (OC)
Colurnn 0 -38 260
38 - 44 260 -+290
44 · 49 290
Injection port 290
Detector 290

Injection 1-3 ¡.tL (depending on the expeeted amount of
sterols in the substanee to be examined) .
Identification 01 peaks Use the ehromatogram obtained
with the referenee solution to identify the peaks due to
eampesterol, stigmasterol, ~-sitosterol and ¡I. 7-stigmastenol.
Identify the peaks due to the sterols in the ehromatogram
obtained with the test solution using the ehromatogram
2014 Appendix XI E V-A319
surface of the liquid by rotating the cylinder about a vertical

Appendix XI axis. Measure the volume occupied by the drug, inc1uding
any adhering mucilage. Carry out 3 tests at the same time.
The swelling index is given by the mean of the 3 tests.
A. Total Solids
The term 'total solids' is applied to the residue obtained
when the prescribed amount of the preparation is dried to D. Foreign Matter
constant weight under the conditions specified below.
APPARATUS
Herbal drugs should be free from moulds, insects and other
A shallow, fiat-bottomed, fianged dish, about 75 mm in animal contamination.
diameter and about 25 mm deep, made of nickel or other
Foreign matter is material consisting of any or all of the
suitable metal of high heat conductivity and low specific heat
following:
and which is not affected byboiling water. Suitable dishes
are described in British Standard 1742: 1951 (Methods for 1) Foreign organs: matter coming from the source plant but
the chemical analysis of condensed milk). not defined as the drug,
2) Foreign elements: matter not coming from the source
METHOD
plant and either of vegetable or mineral origino
Place the quantity stated in the monograph in a tared dish,
evaporate at as low a temperature as possible until the Determination ofForeign Matter
ethanol is removed and heat on a water-bath until the residue
is apparently dry. Transfer to an oven operating without a Weigh 100 g to 500 g ofthe substance to be examined, or
fan and dry to constant weight at 105 0 unless otherwise the minimum quantity prescribed in the monograph, and
stated in the monograph. It may be necessary, for residues of spread it out in a thin layer. Examine for foreign matter by
a hygroscopic nature, to use a dish provided with a well- inspection with the unaided eye or by use of a lens (6 x).
fitting cover and to cool in a desiccator. Separate foreign matter and weigh it and calculate the
percentage presento
81. Ethanol-soluble Extractive

(No Ph. Eur. method) E. Essential Oils in Herbal Drugs
Macerate 5 g of the air-dried drug, coarsely powdered, with (Ph. Eur. method 2.8.12)
100 mL of ethanol of the specified strength in a closed fiask The determination of essential oils in herbal drugs is carried
for 24 hours, shaking frequently during the first 6 hours and out by steam distillation in a special apparatus in the
then allowing to stand for 18 hours. Filter rapidly, taking conditions described below. The distillate is collected in the
precautions against los s of ethanol, evaporate 20 mL of the graduated tube, using xylene to take up the essential oil;
filtrate to dryness in a tared, fiat-bottomed, snallow dish and the aqueous phase is automatically returned to the distillation
dry at 105° to constant weight. Calculate the percentage of fiask.
ethanol-soluble extractive with reference to the air-dried
Apparatus The apparatus comprises the following parts:
drug.
(a) a suitable round-bottomed fiask with a short, ground-
glass neck having an internal diameter of about 29 mm
at the wide end;
82. Water-soluble Extractive (b) a condenser assembly (se e Figure 2.8.12.-1) that c10sely
(No Ph. Eur. method) fits the fiask, the different parts being fused into one
To 5 g of the powdered drug (710) add 200 mL of boiling piece; the glass used has a low coefficient of expansion:
water. Allow to stand for 10 minutes, shaking occasionally. - the stopper K' is vented and the tube K has an orifice of
Allow to cool, dilute to 200 mL with water and filter. diameter about 1 mm that coincides with the ventó
Evaporate 20 mL of the filtrate to dryness on a water-bath. the wide end of the tube K is of ground-glass and has an
Dry the residue in an oven at 100° to 105° for 15 minutes. internal diameter of 10 mm;
- a pear-shaped swelling, J, of 3 mL capacity;
- the tube JL is graduated in 0.01 mL;
C. Swelling Index - the bulb-shaped swelling L has a capacity of about 2 mL;
(Ph. Eur. method 2.8.4) - M is a three-way tapó
The swelling index is the volume in millilitres occupied by - the junction B is at a level 20 mm higher than the
1 gram of a drug, including any adhering mucilage, after it uppermost graduation;
has swollen in an aqueous liquid for 4 h. (c) a suitable heating device, allowing a fine control;
In a 25 mL ground-glass stoppered cylinder graduated over a (d) a vertical support with a horizontal ring covered with
height of 125 ± 5 mm in 0.5 mL divisions, place 1.0 g of insulating material.
the drug, whole or of the degree of comminution prescribed
in the monograph. Unless otherwise prescribed, moisten the
drug with 1.0 mL of alcohol R, add 25 mL of water R and
close the cylinder. Shake vigorously every 10 min for 1 h.
Allow to stand for 3 h. At 90 min after the beginning of the
test, release any large volumes of liquid retained in the layer
of the drug and any partic1es of the drug fioating at the
V-A320 Appendix XI F 2014
150
Mark b
t
=3mL
D
t
Marka
o
L()
o
L()
Figure 2.8.12.-2
Introduce into the fiask the prescribed quantity of the drug

and continue the distillation as described aboye for the time
and at the rate prescribed. Stop the heating and after 10 min
C -l---I'-+-\- read the volume of liquid collected in the graduated tube and
subtract the volume of xylene previously noted.
The difference represents the quantity of essential oil in the
mas s of the drug taken. Calculate the result as millilitres per
kilogram of drug.
When the essential oil is to be used for other analytical
purposes, the water-free mixture of xylene and essential oil
may be recovered as follows: remove the stopper K' and
introduce 0.1 mL of a 1 giL solution of sadium
fiuaresceinate R and 0.5 mL of water R . Lower the mixture of
o
o
~
xylene and essential oil into the bulb-shaped swelling L by
means of the three-way tap, allow to stand for 5 min and
lower the mixture slowly until it just reaches the level of the
tap M. Open the tap anti-clockwise so that the water fiows
out of the connecting tube BM. Wash the tube with
A acetone R and with a little toluene R introduced through the
filling funnel N. Tum the tap anti-c1ockwise in order to
recover the mixture of xylene and essential oil in an
appropriate fiask.
Figure 2.8.12.-1. - Apparatus for the determination of
essential oils in herbal drugs
Dimensions in millimetres F. Continuous Extraction of Drugs
(Na Ph. Eur. methad)
Method Use a thoroughly cleaned apparatus. Carry out the Where the process of maceratian or percalatian is specified in a
assay according to the nature of the drug to be examined. monograph, carry out the following procedures with any
Place the prescribed volume of distillation liquid in the fiask, modification indicated in the monograph.
add a few pieces of porous porcelain and attach the MACERATION
condenser assembly. Introduce water R through the filling Place the solid materials with the whole of the menstruum in
funnel N until it is at the level B. Remove the stopper K' a closed vessel and allow to stand for 7 days, shaking
and introduce the prescribed quantity of xylene R, using a occasionally. Strain, press the marc and mix the liquids
pipette with its tip at the bottom of the tube K. Replace the obtained. ClarifY by subsidence or filtration.
stopper K ' and ensure that the orifice coincides with the
PERCOLATION
vento Reat the liquid in the fiask to boiling and adjust the
distillation rate to 2-3 mUmin, unless otherwise prescribed. Moisten the solid materials with a sufficient quantity of the
menstruum, allow to stand for 4 hours in a well-closed
To determine the rate of distillation, during distillation lower
vessel, pack in a percolator and add sufficient of the
the level of the water by meaÍls of the three-way tap until the
menstruum to saturate the materials. When the liquid begins
meniscus is at the level of the lower mark (a) (see Figure
to drop from the percolator, close the outlet, add sufficient of
2.8.12.-2). Close the tap and measure the time taken for the
the menstruum to leave a layer aboye the drug and allow to
liquid to reach the upper mark (b). Open the tap and
macerate for 24 hours. Allow percolation to proceed slowly
continue the distillation, modifYing the heat to regulate the
until the percolate mea sures about three-quarters of the
distillation rateo Distil for 30 mino Stop the heating and after required volume. Press the marc, mix the expressed liquid
at least 10 min read off the volume of xylene in the
with the percolate and add sufficient of the menstruum to
graduated tube.
produce the required volume. ClarifY by subsidence or
filtration.
Continuous extraction of a drug for the purpose of an assay
consists of percolating the drug with the solvent stated in the
monograph at a temperature approximately that of the
boiling point of the solvento
The apparatus described below, or any similar apparatus,
may be used, provided that it permits the uniform
2014 Appendix XI H V-A321
percolation of the drug and the regular fiow of the vapour of EXTRACTION WITH AN IMMISCIBLE SOLVENT
the solvent around the percolator. After extracting at least three times with the solvent, add to
The apparatus is shown in Fig. llF- l. A is an outer tube of 1 to 2 mL of the next portion 1 to 2 mL of 0.1 M hydrochloric
stout glass; the wider part is about 18 cm long and has an acid, remove the organic solvent by evaporation, transfer the
intemal diameter of 4.8 to 5 cm; the lower end C is about aqueous residue to a test tube and add 0.05 mL of potassium
5 cm long and has an external diameter of about 1.6 cm. B tetraiodomercurate solution or, for solanaceous alkaloids,
is a straight glass tube open at both ends, about 9 cm long 0.05 mL of potassium iodobismuthate solution Rl or, for
and with an external diameter of about 3.8 cm; over its emetine, 0.05 mL of iodinated potassium iodide solution.
lower, fianged end is tied firmly a piece of calico or other Not more than a very faint opalescence is produced.
suitable material. D is a glass coil which supports the margin CONTINUOUS EXTRACTION
of the tube B and prevents ir from resting in contact wirh the After percolating for at least 2 hours, collect 1 to 2 mL of the
outer tube A. The lower end C of the outer tube A is fined effiuent and carry out the procedure described under
by a cork or ground-glass joint to the distillation fiask E, in 'Extraction with an aqueous or alcoholic liquid' or
which a suitable quantity of the solvent has been placed. 'Extraction with an irnmiscible solvent', as appropriate.
The drug to be extracted, previously moistened with the
solvent or subjected to any preliminary treatment required, is
introduced into the inner tube B, which is supported so that
the percolate drops into the outer tube. A pad of absorbent H. Stomata
corton G is placed on the top of the drug, the inner tube is (Ph. Eur. method 2.8.3)
lowered into position and the outer tube connected by means There are several types of stomata (see Figure 2.8.3.-1),
of a suitable cork or ground-glass joint with the tube of a distinguished by the form and arrangement of the
refiux condenser F . The fiask is heated and the extraction surrounding cells:
continued as directed. (1) The anomocytic (irregular-celled) type: the stoma is
surrounded by a varying number of cells in no way
differing from those of the epidermis generally,
(2) The anisocytic (unequal-celled) type: the stoma is usually
surrounded by 3 subsidiary cells, of which one is
markedly smaller than the others,
(3) The diacytic (cross-celled) type: the stoma is
A
accompanied by 2 subsidiary cells, whose common wall
B is at right angles to the guard cells,
(4) The paracytic (parallel-celled) type: the stoma has on
each side one or more subsidiary cells parallel to the
long axis of the pore and guard cells.
Stomatal Index
100 x S
Stomatallndex = E+S
S the number of stomata in a given are a of leaf,

E the number of epidermal cells (including trichomes) in
the same area of leaf.
For each sample of leaf, make not fewer than 10
determinations and calculate the mean.
Fig. llF-l Apparatus for the Contínuous Extraeríon of Drugs
G. Complete Extraction 01 Alkaloids

Complete extraction is indicated by the following tests.
EXTRACTION WITH AN AQUEOUS OR ALCOHOLIC LIQUID
After extracting at least three times with the liquid, add to 3 4
0.1 to 0.2 mL of the next portion, after acidifying with
2M hydrochloric acid if necessary, 0.05 mL of potassium
tetraiodomercurate solution or, for solanaceous alkaloids,
0.05 mL of potassium iodobismuthate solution Rl .
No precipitate or turbidity is produced.
Figure 2.8.3.-1
V-A322 Appendix XI J 2014
J. Ash L. Pesticide Residues

Use Method 1 unless otherwise directed in the monograph. (Ph. Eur. method 2.8.13)
Definitíon For the purposes of the Pharrnacopoeia, a
Method 1 pesticide is any substance or mixture of substances intended
(No Ph. Eur. method) for preventing, destroying or controlling any pest, unwanted
FOR HERBAL DRUGS species of plants or animals causing harrn during or
Incinerate 2 to 3 g of the ground drug in a tared platinum or otherwise interfering with the production, processing,
silica dish at a temperature not exceeding 450° until free storage, transport or marketing of herbal drugs. The item
from carbon, cool and weigh. If a carbon-free ash cannot be inc1udes substances intended for use as growth-regularors,
obtained in this way, exhaust the charred mas s with hot defoliants or desiccants and any substance applied to crops,
water, collect the residue on an ashless filter paper, incinerate either before or after harvest, to protect the commodity from
the residue and filter paper, add the filtrate, evaporate to deterioration during storage and transporto Pesticide residues
dryness and ignite at a temperature not exceeding 450°. can be present and are controlled in herbal drugs and herbal
Calculate the percentage of ash with reference to the air- drug preparations.
dried drug. Limits Unless otherwise indicated in the monograph, the
FOR OTHER SUBSTANCES
herbal drug ro be examined at least complies with the limits
indicated in Table 2.8.13.-1. The limits applying ro
Carry out the aboye method using 1 g, unless otherwise
pesticides that are not listed in Table 2.8.13.-1 and whose
stated. Calculate the percentage of ash.
presence is suspected for any reason comply with the limits
Method 11 (levels) cross referred ro by Regulation (EC) No. 396/2 005,
inc1uding annexes and successive updates. Limits for
(Ph. Eur. method 2.4.16) pesticides that are not listed in Table 2.8 .13.-1 nor in
Reat a silica or platinum crucible to redness for 30 min, European Union texts are calculated using the following
allow to cool in a desiccator and weigh. Unless otherwise expression:
prescribed, evenly distribute 1.00 g of the substance or the
powdered herbal drug to be examined in the crucible. Dry at
ADlxM
100 oC to 105 oC for 1 h and ignite to constant mass in a
muffie fumace at 600 oC ± 25 oC, allowing the crucible to MDDHD X 100
cool in a desiccator after each ignition. Flames should not be
produced at any time during the procedure. If after ADI acceptable daily intake, as published by FAO-
prolonged ignition the ash still contains black particles, take WHO, in milligrams per kilogram of body
up with hot water, filter through an ashless filter paper and mass,
ignite the residue and the filter paper. Combine the filtrate M body mass in kilograms (60 kg),
with the ash, carefully evaporate to dryness and ignite to MDDHD daily dose of the herbal drug, in kilograms.
constant mass . . The limits for pesticides in herbal drug preparations are
calculated using the following expressions:
IfDER ::; 10:
K. Acid-insoluble Ash MRLBDXDER

Use Method I unless otherwise directed in the monograph.
Method 1 IfDER > 10:
(No Ph. Eur. method) ADI x M

Boil the ash for 5 minutes with 25 mi of 2M hydrochloric acid, MDDHP X 100
collect the insoluble matter in a sintered-glass crucible or on
an ashless filter paper, wash with hot water and ignite.
MRL HD maximum residue limit of the pesticide in the
Calculate the pen;entage of acid-insoluble ash with reference
herbal drug as given in Table 2.8.13.-1 or in
to the air-dried drug.
EU texts or calculated using the expression
mentioned aboye;
Method 11
DER drug/extract ratio, i.e. the ratio between the
(Ph. Eur. method 2.8.1) quantity of herbal drug used in the manufacture
Ash insoluble in hydrochloric acid is the residue obtained of a herbal' drug preparation and the quantity of
after extracting the sulfated or total ash with hydrochloric herbal drug preparation obtained;
acid, calculated with reference to 100 g of drug. MDD HP = daily dose of the herbal drug preparation, in
To the crucible containing the residue from the kilograms.
deterrnination of sulfated or total ash, add 15 mL of water R
The competent authority may grant total or parrial
and 10 mL of hydrochloric acid R, cover with a watch-glass,
exemption from the test when the complete hisrory (nature
boil the mixture gently for 10 min and allow to cool. Filter
and quantity of the pesticides used, date of each treatment
through an ashless filter, wash the residue with hot ziJ"ater R
during cultivation and after the harvest) of the treatment of
until the filtra te is neutral, dry, ignite to dull redness, allow
the batch is known and can be checked precisely according
to cool in a desiccaror and weigh. Reheat until the difference
to good agricultural and collection practice (GACP).
between 2 consecutive weighings is not more than 1 mg.
Sampling of herbal drugs Sampling is done according to
the general chapter 2.8.20 Herbal drugs: sampling and sample
preparation.
2014 Appendix XI L V-A323
Table 2.8.13.-1 Table 2.8.13.-1

Substance Limit Substance Limit
(mg¡'kg) (mgjkg)
Acephate 0.1 Methamidophos 0.05
Alachlor 0_05 Methidathion 0.2
Aldrin and dieldrin (sum 01) 0.05 Methoxychlor 0.05
Azinphos-ethyl 0.1 Mirex 0_01
Azinphos-methyl Monocrotophos 0.1
Bromide. inorganic (calculated as bromide ion) 50 Parathion-ethyl and Paraoxon-ethyl (sum 01) 0.5
Bromophos-ethyl 0.05 Parathion-methyl and Paraoxon-methyl (sum of) 0.2
Bromophos-methyl 0.05 Pendimethalin 0.1
Brompropylate 3 Pentachloranisol 0.01
Chlordane (sum of cis-, trans - and oxychlordane) 0.05 Permethrin and isomers (su m 01)
Chlorfenvinphos 0.5 Phosalone 0.1
Chlorpyriphos-ethyl 0.2 Phosmet 0_05
Chlorpyriphos-methyl 0.1 Piperonyl butoxide 3
Chlorthal-dimethyl 0.01 Pirimiphos-ethyl 0.05
Cyfluthrin (sum 01) 0.1 Pirimiphos-methyl (sum of pirimiphos-methyl and 4

N-desethyl-pirimiphos-methyl)
A-Cyhalothrin
Procymidone 0.1
Cypermethrin and isomers (sum 01)
Profenophos 0.1
DDT (sum of o,p'-DDE, p,p'-DDE, o,p'-DDT, p,p'-DDT,
o,p'-TDE and p.p'-TDE)
Prothiophos 0.05
Deltamethrin 0.5 Pyrethrum (sum of cinerin 1, cinerin 11, jasmolin 1, jasmolin 3

11, pyrethrin I and pyrethrin 11)
Diazinon 0.5
Quinalphos 0.05
Dichlofluanid 0.1
Quintozene (sum of quintozene, pentachloraniline and
Diehlorvos methyl penthachlorphenyl sulfide)
S-421 0.02
Dieofol 0.5
Tecnazene 0.05
Dimethoate and omethoate (sum 01) 0.1
Tetradifon 0.3
Dithiocarbamates (expressed as CS2)
Vinclozolin 0.4
Endosulfan (sum of isomers and endosulfan sulfate) 3
Endrin 0.05
Ethion 2
Qualitative and quantitative analysis of pesticide
residues The analytical procedures used are validated
Etrimphos 0_05 (e.g. according to Document N° SANCO/10232/2006).
Fenchlorophos (sum of fenchlorophos and 0.1 In particular, they satisfy the following criteria:
fenchlorophos-oxon)
- the chosen method, especially the purification steps, is
Fenitrothion 0.5
suitable for the combination pesticide residue/substance to
Fenpropathrin 0.03 be examined, and not susceptible to interference trom
Fensulfothion (sum of fensulfothion, fensulfothion-oxon, 0.05
co-extractives;
fensulfothion-oxonsulfon and fensulfothion-sulfon) - natural occurrence of some constituents is considered in
Fenthion (sum of fenthion, fenthion-oxon, 0.05 the interpretation of results (e.g. disulfide from
fenthion-oxon-sulfon, fenthion-oxon-sulfoxid, fenthion-sulfon Cruciferaceae) ;
and fenthion-sulfoxid)
- the concentration of test and reference solutions and the
Fenvalerate 1.5
setting of the apparatus are such that the responses used
Flucytrinate 0.05 for quantification of the pesticide residues are within the
't-Fluvalinate 0.05 dynamic range of the detector. Test solutions containing
pesticide residues at a leve! outside the dynamic range,
Fonophos 0.05
may be diluted within the calibration range, provided that
Heptachlor (sum of heptachlor, cis-heptachlorepoxide and 0.05 the concentration of the matrix in the solution is adjusted
trans-heptachlorepoxide)
in the case where the calibration solutions must be matrix-
Hexachlorbenzene 0.1 matched;
Hexachlorocyclohexane (sum of isomers a-, [3-, O- and €) 0.3 - between 70 per cent to 110 per cent of each pesticide is
Lindan (y-hexachlorocyclohexane) 0.6 recovered;
Malathion and malaoxon (sum 01)
- repeatability of the method: RSD is not greater than the
values indicated in Table 2.8.13.-2;
Mecarbam 0.05
- reproducibility of the method: RSD is not greater than the
Methacriphos 0.05 values indicated in Table 2.8.13.-2.
V-A324 Appendix XI M 2014
Table 2.8.13.-2
Concentration range Repeatability (RSD) Reproducibility (RSD)
N. Bitterness Value
oC the pesticide (per cent) (per cent) (Ph. Eur. method 2.8.15)
(mg/kg)
The bitterness value is the reciprocal of the dilution of a
0.001·0.01 30 60 compound, a liquid or an extract that still has a bitter taste.
> 0.01 . 0.1 20 40 It is determined by comparison with quinine hydrochloride,
> 0.1 - 1 15 30
the bitterness value ofwhich is set at 200000 .
> 1 10 20 Detenmnation of the correction factor

A taste panel comprising at least 6 persons is recommended.
The mouth must be rinsed with water R before tasting.
To correct for individual differences in tasting bitterness
amongst the panel members it is necessary to determine a
M. Tannins in Herbal Drugs correction factor for each panel member.
(Ph. EUI". method 2.8.14) Stock solution Dissolve 0.100 g of quinine hydrochloride R
Carry out all the extraction and dilution opel"ations protected Irom in water R and dilute to 100.0 mL with the same solvento
light. Dilute l.0 mL of this solution to 100.0 mL with water R.
In the case of a herbal drug or a dry extract, to the stated Reference solutions Prepare a series of dilutions by
amount of the powdered drug (I80) (2.9.12) or the extract in placing in a first tube 3.6 mL of the stock solution and
a 250 mL round-bottomed ftask add 150 mL of water R. increasing the volume by 0.2 mL in each subsequent tube to
Heat on a water-bath for 30 mino Cool under running water a total of 5.8 mL; dilute the contents of each tube to
and transfer quantitatively to a 250 mL volumetric ftask. 10.0 mL with water R.
Rinse the round-bottomed ftask and collect the washings in Determine as follows the dilution with the lowest
the volumetric ftask, then dilute to 250.0 mL with water R . concentration that still has a bitter taste. Take 10.0 mL of
Allow the solids to settle and filter the liquid through a filter the weakest solution into the mouth and pass it from side to
paper 125 mm in diameter. Discard the first 50 mL of the side over the back of the tongue for 30 s. Jf the solution is
filtrate. not found to be bitter, spit it out and wait for 1 mino Rinse
In the case of a liquid extract or a tincture, dilute the stated the mouth with water R. After 10 min, use the next dilution
amount of the liquid extract or tincture to 250.0 mL with in order of increasing concentration.
water R . Filter the mixture through a filter paper 125 mm in Calculate the correction factor k for each panel member from
diameter. Discard the first 50 mL of the filtrate . the expression:
Total polyphenoIs Dilute 5.0 mL of the filtrate to
25.0 mL with water R. Mix 2.0 mL of this solution with n
l.0 mL of phosphomolybdotungstic reagent R and 10.0 mL of k = 5.00
water R and dilute to 25 .0 mL with a 290 gIL solution of
sodium carbonate R. After 30 min measure the absorbance n number of millilitres of the stock solution in the
(2. 2.25) at 760 nm (A l), using water Ras the compensation dilution of lowest concentration that is judged to be
liquido bitter.
Polypheno/s not adsorbed by hide powder To 10.0 mL Persons who are unable to taste any bitterness when using
of the filtrate, add 0.10 g of hide powder CRS and shake the reference solution prepared from 5.8 mL of stock
vigorously for 60 mino Filtet and dilute 5.0 mL of the filtrate solution have to beexcluded from the panel.
to 25.0 mL with water R. Mix 2.0 mL of this solution with
Sample preparation
1.0 mL of phosphomolybdotungstic reagent R and 10.0 mL of
Ifnecessary, reduce the sample to a powder (7 10) (2.9.12) .
water R and dilute to 25.0 mL with a 290 gIL solution of
To 1.0 g of sample add 100 mL of boiling water R. Heat on
sodium carbonate R . After 30 min measure the absorbance
a water-bath for 30 min, stirring continuously. Allow to cool
(2.2.25) at 760 nm (A 2 ), using water R as the compensation
and dilute to 100 mL with water R. Shake vigorously and
liquido
filter, discarding the first 2 mL of the filtrate. The filtrate is
Standard Dissolve lmmediately before use 50.0 mg of labelled C-1 and has a dilution factor (DF) of 100.
pyrogallol R in water R and dilute to 100.0 mL with the same
lf liquids have to be examined, 1 mL of the liquid is diluted
solvento Dilute 5.0 mL of the solution to 100.0 mL with
with a suitable solvent to 100 mL and designated C-1.
water R . Mix 2.0 mL of this solution with 1.0 mL of
phosphomolybdotungstic reagent R and 10.0 mL of water R and Determination 01 the bittemess value
dilute to 25 .0 mL with a 290 giL solution of sodium Test solutions:
carbonate R. After 30 min measure the absorbance (2.2.25) at 10.0 mL of C-1 is diluted with water R to (DF = 1000)
760 nm (A 3 ) , using water R as the compensation liquido 100 mL: C-2
Calculate the percenlage content of tannins expressed as 10.0 mL of C-2 is diluted with water R to (DF = 10000)
pyrogallol from the expression: 100 mL: C-3
20.0 mL of C-3 is diluted with water R to (DF = 50000)
62.5 (A~ - A 2 ) m2 100mL: C-3A
10.0 mL of C-3 is diluted with water R to (DF = 100000)
A3 x ml 100 mL: C-4
mass of the sample to be examined, in grams, Starting with dilution C-4 each panel member determines the
mass of pyrogallol, in grams. dilution which still has a bitter taste. This solution is
designated D. Note the DF of solution D is Y.
2014 Appendix XI R V-A325
Starting with solution D prepare the following sequence of acid I at levels equal to or greater than 2 ppm. It may be
dilutions: applied if chromatographic data suggests the presence of
aristolochic acid I.
Solution O (roL) These methods are not designed for inclusion as assay
water R(roL) methods in monographs on those drugs that produce
aristolochic acids as secondary metabolites; for these, a more
sensitive, validated method is required.
Determine the number of millilitres of solution D which,
when diluted ro 10.0 mL with water R, still has a bitter taste Method A: screening test for aristolochic acids
(X) . Thin-layer chromatography (2.2.27).
Calculate the bitterness value for each panel member from Solvent mixture anhydrous formic acid R, water R,
the expression: methanol R (1:9:40 VIV/V).
Test solution To 1.0 g of the powdered herbal drug (710)
y x k ) (2.9.12) add 10.0 mL of the solvent mixture, sonicate for
( X x 0.1
10 min and centrifuge.
Reference solution (a) Disperse an amount of aristolochia
Calculate the bitterness value of the sample ro be examined
HRS corresponding to 0.10 mg of aristolochic acid I in
as the average value for all panel members.
20.0 mL ofthe solvent mixture, sonicate for 10 min and
centrifuge.
Reference solution (b) Dilute 1.0 mL of reference
P. Dry Residue of Extracts solution (a) to 25 .0 mL with methanal R.
(Ph. Eur. method 2.8.16) Plate TLC silica gel FZ54 plate R (2-10 11m).
In a fiat-bottomed dish about 50 mm in diameter and about Mobile phase anhydrous formic acid R, water R, ethyl
30 mm in height, introduce rapidly 2.00 g or 2.0 mL of the acetate R, toluene R (3:3:30:60 V/VIV/V); use the upper layer.
extract to be examined. Evaporate to dryness on a water-bath Application 20 ¡.tL as bands of 8 mm.
and dry in an oven at 100-105 oC for 3 h. Allow to cool in a
Development Over a path of 6 cm.
desiccaror over diphosphorus pentoxide R or anhydrous silica
gel R and weigh. Calculate the result as a mass percentage or Drying In a current of cold air for 5 mino
in grams per litre. Detection Spray with a 100 gIL solution of stannous
chlaride R in dilute hydrochloric acid R until the plate is slightly
wet, heat at 100 oC for 1 min and examine in ultraviolet
a. Loss on Drying of Extracts light at 365 nm.

System suitability
(Ph. Eur. method 2.8.17) - the chromatogram obtained with reference solution (a)
In a fiat-bottomed dish about 50 mm in diameter and about shows 2 greenish-blue zones due to aristolochic acids I
30 mm in height, weigh rapidly 0.50 g of the extract to be and II between RF = 0.35 and RF = 0.55, which may not
examined, finely powdered. Dry in an oven at 100-105 oC be completely separated;
for 3 h. AlIow to cool in a desiccator over diphosphorus
- the chromatogram obtained with reference solution (b)
pentoxide R or anhydrous silica gel R and weigh. Calculate the
shows at least 1 of these zones (corresponding to 2 ppm
result as a mass percentage.
of aristolochic acid I).
Results In the chromatogram obtained with the test
solution no zone is similar in position and fiuorescence to
R. Test for Aristolochic Acids in Herbal any of the zones due to aristolochic acids in the
chromatogram obtained with reference solution (a).
Drugs
Use Method Rl unless otherwise directed in the monograph. If the chromatogram obtained with the test solution shows
any zones similar in position and fiuorescence to any of the
Rl. Test for Aristolochic Acids in Herbal Drugs zones due to aristolochic acids I and II in the chromatogram
(Ph. Eur. method 2.8.21) obtained with reference solution (a), apply method B.
CAUTION: aristolochic acids are very toxic and carcinagenic. Method B: limit test for aristolochic acid 1
Peiform manipulatians under an extraction hood whenever
Liquid chromatography (2.2.29).
possible. Take particular precautions, such as use of a glove box)
when the substance is in dry form because of its electrostatic Solvent mixture acetonitrile R, water R (50:50 V/V) .
properties and the tendency to disperse thraugh the working areas. Test solution Weigh 2.0 g of the powdered herbal drug
Methods A and B are intended to be cross-referenced in (710) (2.9.12) into a 250 mL, brown, screw-cap bottle and
monographs on herbal drugs that, according to add 100.0 mL of the solvent mixture. Stir for 30 min at
chemotaxonomic knowledge, are expected to be free from about 300 r/min and filter through a membrane filter
aristolochic acids, but that may be subject to adulteration or (nominal pore size 0.45 11m).
substitution with plant material containing arisrolochic acids. Reference solution (a) Dissolve the contents of a vial of
Methods A and B are intended to be used in the screening of aristolochic acid 1 CRS in the solvent mixture ro obtain a
herbal drugs for aristolochic acids at the stated limits and will concentration of 0.04 llg/mL of aristolochic acid 1.
usually be complimented by macroscopic andJor microscopic Reference solution (b) Dissolve the contents of a vial of
tests ro exclude plant material containing aristolochic acids. aristolochic acid for system suitabilily CRS (containing
Method C will not be used in specific monographs but is aristolochic acids 1 and II) in the solvent mixture and dilute
provided as a method ro confirm the presence of aristolochic to 20.0 mL with the solvent mixture.
V-A326 Appendix XI R 2014
Column Time Mobile phase A Mobile phase B

- size: 1 = 0.15 m, 0 = 2.1 mm; (min) (per cen! VIl'l (per cen! VIl'l
0·15 70 ~ O 30 ~ lOO
- stationmy phase: octadecylsilyl silica gel for chromatography R
(3.5 ¡.lm); 15·16 O lOO
- temperature: 40 oC. 16· 17 O ~ 70 lOO ~ 30
Mobile phase
- mobile phase A: trifiuoroacetic acid R, water R (0.1:99.9 Flow rate 0.4 mUmin.
VIV);
Injection 20 ¡.tL; inject reference solution (a) twiee, the
- mobile phase B: tnfluoroacetic acid R, acetonitrile R (0.1:99.9 test solution twiee, reference solution (a) twice, then
VIV); reference solution (b) twice.
Detection Mass detector as described below under A or B.
Time Mobile phase A Mobile phase B Adjust the fiow rate, the temperature and the detector
(min) (per cen! VIl'l (per cen! VIl'l settings so as to comply with the system suitability criterion.
0·25 85 ~ 35 15 ~ 65 A. Ion-trap mass spectrometer equipped with an
25·30 35 ~ O 65 ~ lOO electro spray ionisation (ESI) interface and MS n analyser.
30·31 O ~ 85 100 ~ 15 Set the mass spectrometer parameters for the MS 3 mode as
follows:

Parent Isolation width Relative collision
Detection Speetrophotometer at 390 nm. Mode (mi z) (mi z) energy (per cent)
Injection 25 ¡.lL.
359
System suitability MS'
[M + NH,r
2.0 30
- resolution: minimum 3.0 between the peaks due to
aristoloehie aeids I and II in the ehromatogram obtained MS 3 298 2.0 35
with referenee solution (b);
- signal-to-noise ratio: minimum 10 for the peak due to - full scan of product ions: from mIz 80 ro mIz 370;
aristoloehie aeid 1 in the ehromatogram obtained with
- product ion s to be monitored: mIz 252, mIz 268 and mIz
referenee solution (a).
281.
Limit:
System suitability
- the sample eomplies with the test if the ehromatogram
- signal-to-noise ratio: minimum 100 for the monitored
obtained with the test solution shows no peak with the
product ion s in the chromatogram obtained with reference
same retention time as the peak due to aristolochic acid I
solution (a);
in the chromatogram obtained with reference solution (a)
(2 ppm) . - matrix interference test: the average of the 2 ratios of
reference solution (b) is inside the ± 40 per cent interval
Method C: confirmatory test for aristolochic acid 1 of the average of the 2 ratios of reference solution (a);
otherwise it is necessary to adjust the detector settings.
Liquid chromatography (2.2.29) coupled with mass
spectrometry (2.2.43). Results Evaluate the average ratios (252/268 and 281/268)
of the relative intensity of the 3 product ion s of aristolochic
Solvent mixture acetonitrile R, water R (50:50 VIV).
acid 1 in the test solution; evaluate the average of the 2 ratios
Test solution Weigh 2.0 g of the powdered herbal drug of the signals at the retention time of aristolochic acid I in
(710) (2.9.12) intoa 250 mL, brown, screw-cap bottle and reference solution (a); if the average of the 2 ratios of the
add 100.0 mL ofthe solvent mixture. Sonicate for 30 min test solution is within the ± 40 per cent interval of the
and filter through a membrane filter (nominal pore size average of the 2 ratios of reference solution (a), aristolochic
0.45 ¡.lm). acid 1 is present in the test solution.
Reference solution (a) Dissolve the contents of a vial of B. Triple-quadrupole mass spectrometer equipped with an
aristolochic acid 1 CRS in the solvent mixture to obtain a ESI interface and MS n analyser.
concentration of 0 .04 ¡.tg/mL of aristolochic acid I.
Set the mass spectrometer parameters for the MS2 mode as
Reference solution (b) Prepare a solution according to follows:
the instructions supplied with aristolochic acid 1 CRS to
- precursor ion: mIz 359 [M + NH,J +;
obtain a concentration of 0.45 ¡.lg of aristolochic acid 1 in
10.0 mL of the test solution. - product ion s to be monitored: mIz 265, mIz 281 and mIz
296.
Column
System suitability:
- size: l = 0.15 m, 0 = 2.1 mm;
- signal-to-noise ratio: minimum 100 for the monitored
- stationary phase: octadecylsilyl silica gel for chromatography R
product ions in the chromatogram obtained with reference
(3.5 ¡.tm);
solution (a);
- temperature: 40 C.
- matrix interference test: the average of the 2 ratios of
Mobile phase reference solution (b) is inside the ± 40 per eent interval
- mobüe phase A : anhydrous formic acid R, 1 gIL solution of of the average of the 2 ratios of reference solution (a);
ammonium acetate R in water R (0. 1:99.9 VIV); otherwise it is necessary to adjust the detector settings.
- mobile phase B : anhydrous formic acid R, 1 gIL solution of Results Evaluate the average ratios (265/281 and 2961281)
ammonium acetate R in methanol R (0.1:99.9 VIV); of the relative intensity of the 3 product ions of aristoloehic
2014 Appendix XI S V-A327
acid 1 in the test solution; evaluate the average of the 2 ratio s Inject 10 ¡.¡L of solution (2) and allow the chromatography to
of the signals at the retention time of aristolochic acid 1 in proceed for 10 minutes. The test is not valid unless the
reference solution (a); if the average of the 2 ratios of the resolution factor between the peaks corresponding to
test solution is within the ± 40 per cent interval of the aristolochic acid II (retention time about 6 minutes) and
average of the 2 ratios of reference solution (a), aristolochic aristolochic acid 1 (retention time about 7 minutes) is at least
acid 1 is present in the test solution. 3.0. Inject solution (2) six times. The relative standard
deviation of the areas of the peaks is at most 1.5% .
2. Test for Aristolochic Acids I and 11 in Herbal Inject 10 ¡.¡L of solution (1) and allow the chromatography to
Drugs proceed for 30 minutes. In the chromatogram obtained with
(No Ph Eur Method) solution (1) the peaks due to aristolochic acid 1 and
CAUTION Aristolochic acids have been shown to be highly toxic aristolochic acid II are absent.
and carcinogenic. Extraordinary care should be taken in any
procedure in which they are used.
In line with the prohibition of the use of Aristolochia species
in unlicensed herbal medicines in the United Kingdom, a test S. Determination 01 Mycotoxins in Herbal
for absence of aristolochic acids 1 and II in herbal drugs has Drugs
been inc1uded in the British Pharmacopoeia. The limit of
detection has been shown to be 0.00078 mg/mL 1. Determination of Aflatoxin Bl in Herbal Drugs
(approximately 1 ppm of aristolochic acids 1 and II). It is (Ph. Eur. method 2.8.18)
advised that the limit of detection for the system-in-use is CAUTlON: aflatoxins are vely toxic and carcinogenic. Peiform
determined by the analyst. Aristolochic acids 1 and II are not manipulations under an extraction hood whenever possible. Take
confined to the genus Aristolochia. The acids are also particular precautions, such as use of a glove box, when toxins are
reponed as present in certain species of Asarum. in dry form because of their electrostatic properties and the
tendency to disperse through the working areas. Decontamination
Sample preparation
procedures for laboratory wastes of aflatoxins were developed by
Unless otherwise specified in the monograph, weigh 2 g of the International Agency for Research on Cancer (JARC).
the ground herbal drug into a centrifuge tube. Add 10 mL of
Afiatoxins are naturally occurring mycotoxins produced
O.lM sodium hydroxide, shake for at least 2 hours and
mainly by Aspergillus flavus and Aspergillus parasiticus. These
centrifuge the mixture for 10 minutes at approximately
fungi are common and widespread in nature and are most
4000 revolutions per minute. Filter the supernatant layer if
often found when certain grains are grown under conditions
visible partic1es remain in the suspension. Pass a 1.0 mL
of stress such as drought. The mould occurs in soil, decaying
portion of the sample solution with the aid of vacuum
vegetation, hay, and grains undergoing microbial spoilage,
through a solid-phase extraction column of 1 mL capacity
and invades all types of organic substrates whenever and
and containing 30 mg of divinylbenzene and vinylpyrrolidone
wherever the conditions are favourable for its growth.
copolymer for chromatography (30 ¡.¡m) (Waters Oasis HLB,
Favourable conditions inc1ude high moisture content and
30 mg/mL or .Phenomenex StrataX, 30 mg/mL is suitable)
high temperature. At least 13 different types of afiatoxin are
previously washed with 1 mL of methanol, followed by 1 mL
produced in nature and most of these are known to be highly
of water. Wash the column with 1 mL of 0.1M sodium
toxic and carcinogenic. Afiatoxin Bl is considered the most
hydroxide followed by 1 mL of a mixture containing
toxico Herbal drugs that are subject to contamination by
2 volurnes of glacial acetic acid, 28 volumes of water and
afiatoxins are tested by a validated method.
70 volumes of methanol. Elute the sample with 0.25 mL of a
mixture containing 98% of methanol and 2% of concentrated Unless otherwise indicated in the monograph, herbal drugs
ammonia. Evaporate the extract to dryness at 40° under a contain not more than 2 ¡.¡g/kg of afiatoxin Bl'
stream of nitrogen and dissolve in 0.25 mL of methanol. If a The competent authority may also require compliance with a
larger sample volume is required then a larger capacity solid- limit of 4 ~lg/kg for the surn of afiatoxins B l , Bz, G l and G z.
phase extraction column may be used. The method described below is cited as an example of a
Use this as solution (1) for the identification method method that has been shown to be suitable for devil's c1aw
described below. root, ginger and senna pods. Its suitability for other herbal
drugs must be demonstrated or another validated method
Identification used.
Carry out the method for liquid chromatography, Method
Appendix III D, using the following solutions. Prepare
Liquid chromatography (2.2.29).
solution (1) as described aboye under Sample preparation.
Solution (2) contains 0.01% w/v of aristolochic acid BPCRS in Aflatoxins are subject to light degradation. Carry out the
metha11Ol. detennination protected from daylight by using UV-absorbing foil
on windows in combination with subdued light, or curtains or
The chromatographic pro ce dure may be carried out using
blinds in combination with artificiallight (fluorescent tubes are
(a) a stainless steel column (25 cm x 4.6 mm) packed with
acceptable). Protect aflatoxin-containing solutions from daylight.
base-deactivated octacf:ecylsilyl silica gel for chromatography (4
¡.¡m) (Genesis C18 is suitable) maintaining the column Rinse glassware before use with a 10 per cent V/V solution of
temperature at 30°, (b) a mixture of 45 volumes of 0.1 % v/v sulfuric acid R and then rinse carefully with distilled water R
of orthophosphoric acid and 55 volurnes of acetonitrile as the until no more acid is presento
mobile phase with a fiow rate of 1.3 mL per minute and (c) Test solution Use an immunoaffinity column containing
a detection wavelength of 225 nm. The identity of any peaks antibodies against afiatoxin Bl with a capacity of not les s
suspected to be due to arisotolochic acids 1 and II may be than 100 ng of afiatoxin Bl and which gives a recovery of
c1arified by use of the UV spectrum recorded with a diode not less than 80 per cent when a solution of 5 ng of afiatoxin
array detector. Bl in a mixture of 12.5 mL of methanol R and 87.5 mL of
V-A328 Appendix XI S 2014
water R is passed through. Allow the irnrnunoaffinity column Table 2.8.18.-1. - Aflatoxin E l standard solutions
ro reach room temperature. To 5.00 g of the powdered drug Final concentration
Volume oi secondary
(500) (2.9.12) add 100 mL of a mixture of 30 volumes of Standard solution of standard solution
stock solution (~L)
(ng!mL)
water R and 70 volumes of methanol R and extract by
125 0.05
sonication for 30 min. Filter through folded filter papero
Pipette 10.0 mL of the clear filtrate into a 150 mL conical 2 250 0.1
flask. Add 70 mL of water R. Pass 40 mL through the 3 500 0.2
irnrnunoaffinity column at a flow rate of 3 mlJmin (not
4 750 0.3
exceeding 5 mUmin). Wash the column with 2 volumes,
each of 10 mL, of water R at a flow rate not exceeding 5 1000 0.4
5 mlJmin and dry by applying a slight vacuum for 5-10 s or
by passing air through the irnrnunoaffinity column by means
of a syringe for 10 S. Apply 0.5 mL of methanol R ro the Calibration curve Prepare the calibration curve using
column and allow to pass through by gravity. Collect the aflatoxin Bl standard solutions 1 to 5, which cover a range
eluate in a 5 mL volumetric flask. After 1 min, apply a 2 n d equivalent to 1-8 ¡.¡g/kg of aftatoxin Bl in the herbal drug.
portion of 0.5 mL of methanol R. After a further 1 min, Check the plot for linearity. If the content of aflatoxin Bl in
apply a 3rd portion of 0.5 mL of methanol R. Collect most of the sample to be examined is outside of the calibration
the applied elution solvent by pressing air through or range, the test solution must be diluted to an aftatoxin
applying vacuum to the column. Dilute to 5 mL with content that is appropriate for the established calibration
water R and shake well. If the solution is clear it can be used curve.
directly for analysis. Otherwise, pass it through a disposable Column:
filter unit prior to injection. U se a disposable filter unit - size: l = 0.25 m, 0 = 4.6 mm;
(e.g. 0.45 ¡.¡m pore size polytetrafluoroethylene filter) that has - stacionary phase: octadecylsilyl silica gel for chromatography R
been shown not to cause loss of aflatoxin by retention. (5 ¡.¡m).
Aflatoxin B¡ primary stock solution Dissolve afiatoxin Mobile phase:
B 1 R in a mixture of 2 volumes of acetonitrile R and
- mobile phase A (for post-column derivatisation with
98 volumes of toluene R to give a 10 ¡.¡g/mL solution.
photochemical reactor or pyridinium bromide):
To determine the exact concentration of afiatoxin B 1 in the
acetonitrile R, methanol R, water R (2:3:6 VIV/V);
stock solution, record the absorption curve (2.2.25) between
330 nm and 370 nm in quartz cells. - mobile phase B (for post-column derivatisation with
electrochemically derived bromine) : add 0.12 g of
Calculate the aflatoxin Bl mass concentration, in micrograms
potassium bromide R and 350 ~lL of dilute nitric acid R1 per
per millilitre, using the following expression:
litre of mobile phase A.
A x M x 100 Flow rate: 1 mlJmin.
é X l
Detection: Fluorescence detector with a 360 nm excitation
filter and a 420 nm cut-off emission filter, or equivalent.
Recommended settings for adjustable detectors are 365 nm
A absorbance determined at the maximum of the
(excitation wavelength) and 435 nm (emission wavelength).
absorption curve;
M molar mass of aflatoxin Bl (312 g/mol); Injection : 500 ¡.¡L.
molar absorptivity of aflatoxin Bl in the toluene- Post-column derivatisation with pyridinium
acetonitrile mixture (1930 m 2/mol); hydrobromide perbromide (PBPB):
optical path length of the cell (1 cm). - pulseless pump;
Aflatoxin B ¡ secondary stock solution Prepare a - T-piece with zero dead volume;
secondary stock solution containing 100 ng/mL aflatoxin Bl - polytetrafluoroethylene reaction tube; l = 0.45 m,
by diluting aflatoxin Bl primary stock solution with a 0 = 0.5 mm;
mixture of 2 volumes of acetonitrile R and 98 volumes of - mobile phase A;
toluene R . Wrap the flask tightly in aluminium foil and store
it below 4 oC. Before use, do not remove the aluminium foil - post-column derivatisation reagent: dissolve 50 mg of
pyridinium hydrobromide perbromide R in 1000 mL of
until the contents have reached room temperature. If the
water R (store protected from light and use within 4 days);
solution has to be stored for a long period (for example,
1 month), weigh the flask and record the mass before and - flow rate of the derivatisation reagent: 0.4 mUmin.
after each use of the solution. Post-column derivatisation with photochemical reactor
Aflatoxin B ¡ standard solutions Place the volumes of (PHRED)
aflatoxin secondary stock solution indicated in - reactor unit with one 254 nm low pressure mercury UV
Table 2.8 .18.-1 in separate 250 mL volumetric flasks. Pass a bulb (minimum 8 W);
stream of nitro gen through at room temperature until the - polished support plate;
solvent has just evaporated. To each flask, add 75 mL of
- knitted reactor coil: polytetrafluoroethylene tubing knitted
methanol R, allow the aflatoxin Bl to dissolve and dilute to
tightly around the UV bulb, l = 25 m, 0 = 0.25 mm,
nominal void volume 1.25 mL;
- exposure time: 2 min;
- mobile phase A:
2014 Appendix XI S V-A329
Post-colurnn derivatisation with electrochernically Test solution Use an immunoaffinity column containing
generated brornine (KOBRA): antibodies against ochratoxin A with a capacity of not less
- KOBRA-cell: electrochemical cell that genera tes a reactive than 100 ng of ochratoxin A and which gives a recovery of
form of bromine for derivatisation of aflatoxins, resulting not les s than 70 per cent. Allow the immunoaffinity column
in enhanced fluorescence; available from various to reach room temperature.
commercial suppliers; To 2.00 g of the powdered drug (250) (2.9.12) add 80 mL
- Derivation direct-current supply in series with the of a 30 gIL solution of sodium hydrogen carbonate R and
KOBRA-cell, providing a constant current of about extract by sonication for 30 min (change water of ultrasonic
100 ¡lA; bath after 15 min). Cool to room temperature and dilute to
- polytetrafluoroethylene reaction tube, 1 = O.12 m, 100.0 mL (VI) with the same solution. Centrifuge .
Mix thoroughly 5.0 mL (TI¡) of the clear supernatant with
0= 0.25 mm;
30 mL buffer solution pH 7.4 R and pass the whole solution
- mobile phase B. through the immunoaffinity column at a flow rate of
Elution order aflaroxin G 2 , aflaroxin G I, aflaroxin B2 , 3 mL/min (do not exceed 5 mL/min). Wash the column first
aflaroxin BI' with 10 mL buffer solution pH 7.4 R then with 2 quantities,
Calculation calcula te the calibration curve y = ax + b, each of 10 mL, of water R at a flow rate not exceeding
with aflatoxin Bl concentration (ng/mL) on the x-axis and 5 mL/min and dry by applying a slight vacuum for 5-10 s or
the signal (S) on the y-axis. The aflatoxin Bl concentration by passing air through the immunoaffinity column by means
(C) in a measured solution is equal to S-b of a syringe for 10 S. Apply 0.5 mL of methanol R to the
a
Calculate the aflaroxin Bl content of the herbal drug, in column and allow to pass through by gravity.
nanograms per gram, using the following expression: Collect the eluate in a 4 mL glass vial. After 30 s, apply a 2nd
quantity of 0.5 mL of methanol R and allow to pass through
the column by gravity into the same glass vial. After a further
30 s, repeat with a 3rd portion of 0.5 mL of melhanol R.
Collect any solvent retained on the column by pressing air
m mas s of the herbal drug taken for analysis, in grams; through or applying vacuum to the column. Evaporate the
VI volume of the solvent used for extraction, in combined eluates completely to dryness using a thermal
millilitres; block with a nitrogen blanket (40 oC). Dissolve the residue in
TI¡ aliquot taken for immunoaffinity clean-up, in 0.5 mL (V2 ) of solution A. If the solution is clear it can be
millili tres; used directly for analysis. Otherwise, pass it through a
V2 final volume of solution after elution from the disposable filter unit prior to injection. Use a disposable filter
immunoaffinity column and dilution, in millilitres; unit (e.g. 0.45 ¡lm pore size polytetrafluoroethylene filter)
C measured aflatoxin BI concentration of the test that has been shown not ro cause loss of ochraroxin A by
solution, in nanograms per millilitre. retention.
The presence of aflatoxin Bl may be confirmed by recording Ochratoxin A prirnary stock solution Dilute 1.0 mL of
the chromatogram without post-column derivatisation, which ochratoxin A solution R to 100.0 mL with melhanol R and
leads to a large decrease (greater than 10-fold) in the shake thoroughly.
response due to aflatoxin Bl' Ochratoxin A secondary stock solution Dilute 10.0 mL
of ochratoxin A primary stock solution to 100.0 mL with
2. DetennÍnation of Ochratoxin A in Herbal Drugs metharlOl R and shake thoroughly.
(Ph. Eur. method 2.8.22) Ochratoxin A standard solutions Place the volumes of
CAUTION: ochratoxin A is nephrotoxic and nephrocarcinogenic. ochratoxin A primary stock solution or ochratoxin A
Perform manipulations under an extraction hood. Take particular secondary stock solution indicated in Table 2.8.22.-1 into
precautions, such as use of a glove box, when toxins are in dry separate flasks and make up to 50.0 mL with solution A.
form because of their electrostatic propenies and the tendency to
disperse through the working are as. Decontamination procedures Table 2.8.22.-1. - Ochratoxin A standard solutions
for laboratory glassware containing ochratoxin A are necessary Standard solution Volume oC ochratoxin A Final concentration
(see appendix) . primary stock solution oí ochratoxin A in
(IIL) standard solution
Herbal drugs that are subject to contamination by ochratoxin (ng/mL)
A are tested by a validated method. 5000 50
The method described below is cited as an example of a 2500 25

method that has been shown to be suitable for liquorice
3 1000 10
extract and liquorice root. lts suitability for other herbal
drugs must be demonstrated or another validated method 4 500 5
used. 5 250 2.5
Standard solution Volume oí ochratoxin A Final concentration

METHOD secondary stock of ochratoxin A in
Liquid chromatography (2.2.29). solution (IIL) standard solution
(ng/mL)
Use brown glassware that is free from detergent residues. 6 500 0.5
If necessary rinse glassware before use with a 10 per cent V/V
7 100 0.1
solution of sulfuric acid R and then rinse carefully with distilled
water R until no more acid is presento
Solution A Mix 80 volumes of water R, previously adjusted Calibration curve Prepare the calibration curve using
to pH 2.3 with anhydrous formic acid R, and 20 volumes of ochratoxin A standard solutions 1 to 7, which cover a range
acetonitrile R. equivalent ro 0.5-250 ¡lg/kg of ochratoxin A in the herbal
V-A330 Appendix XI T 2014
drug. Check the plot for linearity. If the content of

ochratoxin A in the sample to be examined is outside of the
T. Herbal Drugs: Sampling and Sample
calibration range, the test solution must be diluted to an Preparation
ochratoxin A content that is appropriate for the established
calibration curve.
In order to reduce the effect of sampling in qualitative and
Column: quantitative analysis, it is necessary to ensure that the
- size: 1 = 0.15 m, 0 = 4.6 mm; composition of the sample used is representative of the batch
- stationary phase: octadecylsilyl silica gel for of material being examined. The following procedures are the
chromatography R (5 ¡.tm); mínimum considered applicable for herbal drugs. NOTE:
- temperature: 45 oC. other procedures may be used if they can be demonstrated to
produce representative batch samples.
Mobile phase:
- mobile phase A: water R adjusted to pH 2.3 with phosphoric Bulk Sample
acid R;
Where external examination of containers, markings and
- mobile phase B: acetonitrile R; labels of a batch indicate that it can be considered to be
homogeneous, sample the number of randomly selected
Time Mobile phase A Moblle pbase B containers indicated below. Where a batch cannot be
(min) (percent VI!? (percent VI!? considered to be homogeneous, divide it into sub-batches
0 -30 80 -; 40 20 -; 60 that are as homogeneous as possible, then sample each sub-
30 - 35 40 -; 20 60 -; 80 batch as a homogeneous batch using, as a minimum, the
number of randomly selected containers indicated below.
35·37 20 80
37 - 40 20 -; 80 80 -; 20
Number of conlainers lo
Number of containers in batch (N)
be sampled (n)
Flow rate 0.8 mUmin. 1-3 aJl
Detection Fluorescence detector; recommended settings >3 n" =..fN +1
for adjustable detectors are 330 nm (excitation wavelength)
" round n up to the next integer
and 460 nm (emission wavelength).
lnjection 20 ¡.tL.
Take one sample from each container to be sampled.
Calculation Calculate the calibration curve y = ax + b,
The sample is taken from the upper, middle or lower section
with ochratoxin A concentration (in nanograms per millilitre)
of the container, such that the samples taken are
on the x-axis and the signal (S) on the y-axis.
representative of different parts of the containers. In the case
The ochratoxin A concentration (C) in a measured solution
of large bales or bags, samples must be taken from a depth of
is equal to S-b
a at least 10 cm. The mas s of the material taken from each
Calculate the ochratoxin A content of the herbal drug, in container is such that the total mas s of the bulk sample
nanograms per gram, using the following express ion: complies wit4 the following values.
Minimum mass of samples as a

Mass of herbal drug in the
percentage of Ihe mass of the
batch (kg)
batch of herbal drug
m mass of the herbal drug used to prepare the test < 50 LOO"
solution, in grams;
50 - 100 0.50
volume of dilution, in millilitres;
aliquot taken for immunoaffinity clean-up, in > 100 - 250 0.25
millilitres; > 250·500 0.20
volume in which the residue is taken up, in millilitres;
> 500 -1000 0.18
measured ochratoxin A concentration of the test
solution, in nanograms per millilitre. > 1000·2500 0.15
> 2500 - 5000 0.10
Appendix: Decontamination procedures for
laboratory glassware > 5000 - 10 000 0.08
Rinse glassware with methanol R and decontaminate by > 10 000 - 25 000 0.05
irnmersion in strong sodium hypochlorite solution R for at least NOTE : if the mass of the batch is greater than 25 000 kg, it is divided
2 h, then wash thoroughly with water. into sub-batches, and the procedure is applied to each sub-batch as
though it were a homogeneous batch.
" subject to a minimum total mass of 125 g for the bulk sample; if this
minimum requirement represents more than 10.0 per cent of the mass of
herbal drug in the batch, the whole batch may be used as the sample.
Prepare the bulk sample by combining and thoroughly

mixing the samples taken from each of the randomly selected
containers (se e Table 2.8.20.-1).
2014 Appendix XI U V-A331
Table 2.8.20.-1. - Operation ofthe sampling procedure in order to obtain the prescribed bulk sample
Mass of herbal
drug in container 0.5 1 5
(kg)
Total mass of No.of No.of Total mass No.of No.of Total mass No.of No.of Total mass
herbal drug in containers containers to of samples containers containers of samples containers containers to of samples
the batch (kg) in batch be sampled (g) in batch to be sampled (g) in batch be sampled (g)
-
0.5 1 1 125
1 2 2 125 1 1 125
5 10 5 125 5 4 125 1 1 125
10 20 6 125 10 5 125 2 2 125
25 - - -
25 6 250 5 4 250
100 - - -
100 11 500 20 6 500
250 - - - - -
50 9 625
500 - - - - 100 11 1000
Mass of herbal
drug in container 25 125 500
(kll)
Total mass of No.of No.of Total mass No.of No.of Total mass No.of No.of Total mass
herbal drug in containers conlainers lo of samples containers containers of samples containers containers to of samples
the balch (kg) in batch be sampled (g) in balch lo be sampled (g) in batch be sampled (g)
25 1 1 250 - -
100 4 3 500 -
250 10 5 625 2 2 625 -
500 20 6 1000 4 3 1000 1 1 1000
1000 40 8 1800 8 4 1800 2 2 1800
2000 80 10 3000 16 5 3000 4 3 3000
3000 120 12 3000 24 6 3000 6 4 3000
5000 200 16 5000 40 8 5000 10 5 5000
10000 400 21 8000 80 10 8000 20 6 8000
25000 800 30 12500 160 14 12500 40 8 12500
Type of berbal drug Minimum weighl of lesl sample the sieve must not be more than 10 per cent of the total
Roots, rhizomes, bark, herbs 500 g or mass of whole sample if
mass of the milled sample, of which not more than
bulk sample is less than 500 g 2 per cent of the total mass of the milled sample may be of a
Leaves, flowers, seeds, fruits 250 g or mass of whole sample if partic1e size greater than 1.5 mm or 1.5 times the specified
bulk sample is less than 250 g partic1e size in the monograph. If these conditions are met,
Broken or fragmented drugs (where 125 g the sample and residue are to be well mixed to form the test
average mass of the pieces is less
than 0.5 g)
sample for analysis.
NOTE: quartering consists of placing the bulk sample,
In those cases where these requirements are not met, the test
thoroughly mixed, as a level and square-shaped heap and sample for analysis is composed of the 2 parts measured
dividing it diagonally into 4 equal parts. 2 opposite quarters separately. Therefore, the quantity required for each analysis
are retained and carefully remixed. The process is repeated is derived by weighing proportional quantities of the powder
as necessary until the required minimum mass is obtained and the residue.
for the test sample. NOTE: for determinacion of microscopic characters, a portian of
the m¡lled test sample is re-milled through a 0.355 mm screen.
Test sample
Unless otherwise prescribed in the monograph, prepare the
test sample as follows.
U. Microscopic Examination of Herbal
Reduce the size of the bulk sample by quartering (see Note
below) or by any other method that produces a Drugs
homogeneous sample, making sure that each retained portion (Ph. Eur. method 2.8.23)
remains representative of the whole, until the minimum The microscopic examination of herbal drugs is carried out
retained quantity complies with the following conditions. on the powdered drug (355) (2.9.12) un les s otherwise
Mili the test sample in a single pass through a 1 mm screen prescribed in the monograph.
or the screen size specified in the monograph. The use of a Chloral hydrate solution R is the most commonly prescribed
milling machine is recommended. reagent. However, certain features are not visible or not easily
Pass the milled sample through a 1 mm standard sieve or the seen after mounting in this reagent. In this case, other
sieve specified in the monograph. The residue retained on reagents are used, for example, a 50 per cent V/V solution of
V-A332 Appendix XI U 2014
glyeerol R, which makes it possible to visualise starch MOUNTING IN RUTHENIUM RED SOLUTION
granules. It may also be necessary to prescribe specific Place 2 drops of ruthenium red solution R on a glass
reagems in a monograph, for example: laetie reagent R which microscope slide. Disperse a very small quamity of the
is used to show the presence of various features, 10 per cem powdered drug in the liquid and cover the preparation with a
VIV alcoholic solution of phloroglucinol R and hydroehloric cover slip. After about 1 minute, allow a drop of distilled
acid R, which are used to idemify the presence of lignin in water R 10 be taken up between the slide and the cover slip.
cells or tissues, ruthenium red solution R, which is used to Examine under a microscope. The mucilage stains violet red.
show the presence of mucilage in cells or glyeerol R used to
show the presence of starch and inulin.
Examination under polarised light (between crossed nicol
prisms) is used to idemify starch granules (black cross
phenomenon), calcium oxalate crystals (refringence) or
Iignified structures.
MOUNTING IN CHLORAL HYDRATE

SOLUTION
Place 2-3 drops of ehloral hydrate solution R on a glass
microscope slide. Disperse a very small quamity of the
powdered drug in the liquid and cover the preparation with a
cover slip. Heat the preparation very gently to boiling on a
hot plate or a micro gas burner. Maimain gentle boiling for a
short time. Make sure that the quamity of mouming fluid is
sufficiem. If necessary, add more fluid using a tapered glass
pipette. Allow to cool and then examine under a microscope.
Repeat the heating until the starch granules and the water-
soluble comems of the cells are no longer visible. Examine
under a microscope.
Chloral hydrate tends to crystallise as long needles. To avoid
this, proceed as follows: after heating, remove the cover slip;
to the preparation add 1 drop of a 10 per cem VIV mixture
of ehloral hydrate solution R in glyeerol R; place a clean cover -
slip on the preparation; examine under a microscope.
MOUNTING IN A 50 PER CENT VIV SOLUTION

OFGLYCEROL
Place 2 drops of a 50 per cent VIV solution of glyeerol R on a
glass microscope slide. Disperse a very small quamity of the
powdered drug in the liquid and cover the preparation with a
cover slip. Examine under a microscope.
MOUNTING IN A 10 PER CENT VIV ALCOHOLlC

SOLUTION OF PHLOROGLUCINOL ANO
HYDROCHLORIC ACm
Place a very small quamiry of the powdered drug on a glass
microscope slide. Add 1-2 drops of a 10 per cem VIV
alcoholic solution of phloroglucinol R. Mix and allow the
solvent 10 evaporate almost completely. Add 1-2 drops of
hydroehlorie acid R and cover the preparation with a cover
slip. Examine immediately under a microscope. The red
colour indica tes the presence of lignin.
MOUNTING IN LACTIC REAGENT

Place 2-3 drops of lactie reagent Ron a glass microscope
slide. Disperse a very small quamity of the powdered drug in
the liquid and cover the preparation with a cover slip. Heat
the preparation very gently to boiling. Maimain gentle boiling
for a short time. Make sure that the quamity of mounting
fluid is sufficient. If necessary, add more fluid using a tapered
glass pipette. Allow to cool and then examine under a
microscope. Lignified structures stain bright yellow;
structures comaining cellulose remain colourless. Starch
granules stain more or les s violet; certain secretions (e.g.,
essential oils, resins, oleoresins) sta in orange and cork stains
red.
2014 Appendix XII A V-A333
assembly conforms to the dimensions shown in Figure

Appendix XII 2.9.1.-1.
Discs The use of discs is permitted only where specified or
allowed. Each tube is provided with a cylindrical disc
A. Disintegration 9.5 ± 0.15 mm thick and 20.7 ± 0.15 mm in diameter.
1. Disintegration Test for Tablets and Capsules l The disc is made of a suitable, transparent plastic material
having a specific gravity of 1.18-1.20. 5 parallel 2 ± 0.1 mm
(Ph. Eur. mechad 2.9.1) holes extend between the ends of the cylinder. One of the
This test is provided to determine whether tablets or capsules holes is centered on the cylindrical axis. The other holes are
disintegrate within the prescribed time when placed in a centered 6 ± 0.2 mm from the axis on imaginary lines
liquid medium under the experimental conditions presented perpendicular to the axis and parallel to each other.
below. 4 identical trapezoidal-shaped planes are cut into the wall of
For the purposes of this test, disintegration does not imply the cylinder, nearly perpendicular ro the ends of the cylinder.
complete dissolution of the unit or even of its active The trapezoidal shape is symmetrical; its parallel sides
constituent. Complete disintegration is defined as that state coincide with the ends of the cylinder and are parallel ro an
in which any residue of the unit, except fragrnents of imaginary line connecting the centres of 2 adjacent holes
insoluble coating or capsule shell, remaining on the screen of 6 mm from the cylindrical axis. The parallel side of the
the test apparatus or adhering ro the lower surface of the trapezoid on the botrom of the cylinder has a length of
discs, if used, is a soft mass having no palpably firm coreo 1.6 ± 0.1 mm and its bottom edges lie at a depth of
Use apparatus A for tablets and capsules that are not greater 1.5 mm ro 1.8 mm from the cylinder's circumference.
than 18 mm long. For larger tablets or capsules use The parallel side of the trapezoid on the top of the cylinder
apparatus B. has a length of 9.4 ± 0.2 mm and its centre lies at a depth
of 2.6 ± 0.1 mm fram the cylinder's circumference.
Test A - Tablets and capsules of normal size All surfaces of the disc are smooth.
Apparatus The apparatus consists of a basket-rack If the use of discs is specified, add a disc ro each tube and
assembly, a 1 litre, low-form beaker, 149 ± 11 mm in opera te the apparatus as directed under Procedure. The discs
height and having an inside diameter of 106 ± 9 mm for conform ro the dimensions shown in Figure 2.9.1.-1.
the immersion fluid, a thermostatic arrangement for heating The use of automatic detection employing modified discs is
the fluid between 35 oC and 39 oC, and a device for raising permitted where the use of discs is specified or allowed. Such
and lowering the basket in the irnmersion fluid at a constant discs must comply with the requirements of densiry and
frequency rate between 29 and 32 cyeles per minute, dimension given in this chapter.
through a distance of 55 ± 2 mm. The volume of the fluid Procedure Place 1 dosage unit in each of the 6 tubes of
in the vessel is such that at the highest point of the upward the basket and, if prescribed, add a disco Operate the
stroke the wire mesh remains at least 15 mm below the apparatus using the specified medium, maintained at
surface of the fluid, and descends ro not less than 25 mm 37 ± 2 oC, as the irnmersion fluid . At the end of the
from the bottom of the vessel on the downward stroke. specified time, lift the basket from the fluid and observe the
At no time should the top of the basket-rack assembly dosage units: all of the dosage units have disintegrated
become submerged. The time required for the upward stroke completely. If 1 or 2 dosage units fail to disintegrate, repeat
is equal ro the time required for the downward stroke, and the test on 12 additional dosage units. The requirements of
the change in stroke direction is a smooth transition, rather the test are met if not less than 16 of the 18 dosage units
than an abrupt reversal of motion. The basket-rack assembly tested have disintegrated.
moves vertically along its axis. There is no appreciable
horizontal motion or movement of the axis from the vertical. Test B - Large tablets and large capsules
Basket-rack assembZy The basket-rack assembly consists Apparatus The main part of the apparatus (Figure
of 6 open-ended transparent tubes, each 77.5 ± 2.5 mm 2.9.1.-2.) is a rigid basket-rack assembly supporting 3
long and having an inside diameter of 21.85 ± 1.15 mm cylindrical transparent tubes 77.5 ± 2.5 mm long, 33.0 mm
and a wall 1.9 ± 0.9 mm thick; the tubes are held in a ± 0.5 mm in internal diameter, and with a wall thickness of
vertical position by 2 plates, each 90 ± 2 mm in diameter 2.5 ± 0.5 mm. Each tube is provided with a cylindrical disc
and 6.75 ± 1.75 mm in thickness, with 6 holes, each 31.4 ± 0.13 mm in diameter and 15.3 ± 0.15 mm thick,
24 ± 2 mm in diameter, equidistant from the centre of the made of transparent plastic with a relative density of
plate and equally spaced from one another. Attached to the 1.18-1.20. Each disc is pierced by 7 holes, each
under surface of the lower plate is a woven stainless steel 3.15 ± 0.1 mm in diameter, 1 in the centre and the other 6
wire eloth, which has a plain square weave with spaced equally on a cirele of radius 4.2 mm fram the centre
2.0 ± 0.2 mm mesh apertures and with a wire diameter of of the disco The tubes are held vertically by 2 separate and
0.615 ± 0.045 mm. The parts ofthe apparatus are superimposed rigid plastic plates 97 mm in diameter and
assembled and rigidly held by means of 3 bolts passing 9 mm thick, with 3 holes. The holes are equidistant from the
through the 2 plates. A suitable means is provided to centre of the plate and equally spaced. Attached ro the under
suspend the basket-rack assembly from the raising and side of the lower plate is a piece of woven gauze made from
lowering device using a point on its axis. stainless steel wire 0.63 ± 0.03 mm in diameter and having
The design of the basket-rack assembly may be varied mesh apertures of 2.0 ± 0.2 mm. The plates are held rigidly
somewhat provided the specifications for the glass tubes and in position and 77.5 mm apart by vertical metal rods at the
the screen mesh size are maintained. The basket-rack periphery. A metal rod is also fixed ro the centre of the
upper plate ro enable the assembly to be attached ro a
mechanical device capable of raising and lowering it
1 This Chapter has undergol1e pharmacopoeial harnlOl1isation. See chapter smoothly at a constant frequency of between 29 and
5.8. Pharmacopoeial harmonisation. 32 cyeles per minute, through a distance of 55 ± 2 mm.
V -A334 Appendix XII A 2014
Basket-rack
assembly
1.9 ± 0.9 Disc
10
~
10
,....
u:i
2.6 ± 0.1
Top
vlew
N
+1 9.4 ± 0.2
~
,....
,....
Side
view
~
N Botlom
+1
view
"'"
N
Figure 2.9.1.-1. - Disintegration apparatus A
The assembly is suspended in the specified liquid medium in the surface of the liquid. A suitable device maintains the
a suitable vessel, preferably a 1 litre beaker. The volume of temperature of the liquid at 35-39 oC.
the liquid is such that when the assembly is in the highest The design of the basket-rack assembly may be varied
position the wire mesh is at least 15 mm below the surface of provided the specifications for the tubes and wire mesh are
the liquid, and when the assembly is in the lowest position maintained.
the wire mesh is at least 25 mm aboye the bottom of the
beaker and the upper open ends of the tubes remain aboye
2014 Appendix XII A V-A335
~
o
+1
Ltl 3.15±O.1
N
~
o
+1
o --1
C'"i
C')
® 31.4±O.13
,.r
•
Ltl
c:i
+1
C')
tri
Figure 2.9.L-2. - Disintegration apparatus B

Method Test 6 tablets or capsules either by using 2 Disintegration is considered to be achieved when:
basket-rack assemblies in parallel or by repeating the a) dissolution is complete,
procedure. In each of the 3 tubes, place 1 tablet or capsule b) the components of the suppository or pessary have
and, if prescribed, add a disc; suspend the assembly in the separated: melted fatty substances collect on the surface
beaker containing the specified liquido Operate the apparatus
of the liquid, insoluble powders fall to the bottom and
for the prescribed period, withdraw the assembly and soluble components dissolve, depending on the type of
examine the state of the tablets or capsules. To pass the test, preparation, the components may be distributed in one
all 6 of the tablets or capsules must have disintegrated. or more of these ways,
2. Disintegration Test for Suppositories and c) there is softening of the sample that may be
Pessaries accompanied by appreciable change of shape without
complete separation of the components, the softening is
such that the suppository or pessary no longer has a
The disintegration test determines whether the suppositories solid core offering resistan ce to pressure of a glass rod,
or pessaries soften or disintegrate within the prescribed time
when placed in a liquid medium in the experimental d) rupture of the gelatin shell of rectal or vaginal capsules
conditions described below. occurs allowirig release of the contents,
e) no residue remains on the perforated disc or if a residue
remains, it consists only of a soft or frothy mass having
V-A336 Appendix XII B 2014
no solid core offering resistance to pressure of a glass samples after the period prescribed in the monograph.
rod (vaginal tablets). To pass the test all the samples must have disintegrated.
Apparatus METHOD OF OPERATION FOR VAGINAL TABLETS
The apparatus (Figure 2.9 .2.-1) consists of a sleeve of glass Use the apparatus described aboye, arranged so as to rest on
or suitable transparent plastic, of appropriate thickness, to the hooks (see Figure 2.9 .2.-2) . Place it in a beaker of
the interior of which is attached by means of three hooks a suitable diameter containing water maintained at 36-37 oC
metal device consisting of two perforated stainless metal discs with the level just below the upper perforated disco Using a
each containing 39 holes 4 mm in diameter; the diameter of pipette, adjust the leve! with water at 36-37 oC until a
the discs is similar to that of the interior of the sleeve; uniform film covers the perforations of the discoUse three
the discs are about 30 mm aparto The test is carried out vaginal tablets. Place each one on the upper plate of an
using three such apparatuses each containing a single sample. apparatus and cover the latter with a glass plate to maintain
Each apparatus is placed in a beaker with a capacity of at appropriate conditions of humidity. Examine the state of the
least 4 L filIed with water maintained at 36-37 oC, unless samples after the period prescribed in the monograph.
otherwise prescribed. The apparatuses may also be placed To pass the test aII the samples must have disintegrated.
together in a vessel with a capacity of at least 12 L.
The beaker is fitted with a slow stirrer and a device that wiII
hold the cylinders verticalIy not less than 90 mm below the
surface of the water and alIow them to be inverted without iA
emerging from the water.
o
lO
oC")
A. glass plate D. water

B. vaginal tablet E. dish, beaker
C. water surface
Figure 2.9.2.-2.
Monographs 01 the British Pharmacopoeia

The foIlowing additional points apply to monographs of the
British Pharmacopoeia.
ACCEPTANCE CRITERIA
For moulded suppositories, disintegration occurs in not more
than 30 minutes for fat-based suppositories and in not more
than 60 minutes for water-soluble suppositories, unless
otherwise justified and authorised.
For moulded pessaries, disintegration occurs in not more
than 60 minutes unless otherwise justified and authorised.
24 For rectal capsules and vaginal tablets and capsules,
36 disintegration occurs in not more than 30 minutes.
Figure 2.9.2.-1. - Apparatus for disintegration of suppositories B. Dissolution

and pessaries
1. Dissolution Test for Tablets and Capsules
Dimensions in millimetres (Dissolution Test for Solid Dosage Fonns)
Method This test is provided to determine compliance with the
Use three suppositories or pessaries. Place each one on the dissolution requirements for solid dosage forms administered
lower disc of a device, place the latter in the sleeve and oraIly. In this chapter, a dosage unit is defined as 1 tablet or
secure. lnven the apparatuses every 10 mino Examine the 1 capsule or the amount specified.
2014 Appendix XII B V-A337
APPARATUS
Apparatus 1 (Basket apparatus) The assembly consists
of the foIlowing: a vessel, which may be covered, made of
glass or other inert, transparent material! ; a motor; a drive
shaft; and a cylindrical basket (stirring element). The vessel
is partialIy irnmersed in a suitable water-bath of any
convenient size or heated by a suitable device such as a
6.3 to 6.5
heating jacket. The water-bath or heating device permits or9.4 to 10.1
maintaining the temperature inside the vessel at 37 ± 0.5 cC
during the test and keeping the dissolution medium in
constant, smooth motion. No part of the assembly, incIuding LO
o
the environment in which the assembly is placed, contributes Vent hole +1
2.0 ± 0.5
significant motion, agitation, or vibration beyond that due to
the smoothly rotating stirri,ng element. Apparatus that
permits observation of the preparation and stirring element Retention spring
during the test is preferable. The vessel is cylindrical, with a with 3 tangs
hemispherical bottom and a capacity of 1 litre. Its height is on 120' centers
I• •I
160-210 mm and its inside diameter is 98-106 mm. Its sides Clear opening
are ftanged at the topo A fitted cover may be used to retard 20.2 ± 1.0
evaporation2 . The shaft is positioned so that its axis is not
more than 2 mm at any point from the vertical axis of the
vessel and rotates smoothly and without significant wobble Screen O.D
that could affect the results. A speed-regulating device is 22.2 ± 1.0
used that aIlows the shaft rotation speed to be selected and
el el
maintained at a specified rate, within ± 4 per cent. M
+1 +1
Shaft and basket components of the stirring element are o o
t-- ~
fabricated of stainless steel, type 316 or equivalent, to the N M
specifications shown in Figure 2.9.3.-l.
A basket having a gold coating of about 2.5 ¡.lm (0.0001
inch) thick may be used. The dosage unit is placed in a dry
A
basket at the beginning of each test. The distance between
the inside bottom of the vessel and the bottom of the basket
is maintained at 25 ± 2 mm during the test.
Apparatus 2 (Paddle apparatus) Use the assembly from
Apparatus 1, except that a paddle formed from a blade and
a shaft is used as the stirring element. The shaft is positioned o o
so that its axis is not more than 2 mm from the vertical axis M
¡¡ +1
of the vessel, at any point, and rota tes smoothly without "! o
o .n
significant wobble that could affect the results. The vertical N N
center line of the blade passes through the axis of the shaft
so that the bottom of the blade is ftush with the bottom of
the shaft. The paddle conforms to the specifications shown
in Figure 2.9.3.-2. The distance of 25 ± 2 mm between the 1) Screen with welded seam: 0.22·0.31 mm wire
bottom of the blade and the inside bottom of the vessel is diameter with wire opening of 0.36-0.44 mm. After
welding the screen may be slighty altered.
maintained during the test. The metaIlic or suitably inert,
2) Maximum allowable runout at "A" is 1.0 mm
rigid blade and shaft comprise a single entity. A suitable two- when the part is rotated on center line axis with
part detachable design may be used provided the assembly basket mounted.
remains firmly engaged during the test. The paddle blade Figure 2.9.3.-1. - Apparatus 1, Basket stirring element
and shaft may be coated with a suitable coating so as to Dimensions in millimetres
make them inert. The dosage unit is aIlowed to sink to the
bottom of the vessel before rotation of the blade is started.
Apparatus 3 (Reciprocating cylinder) The assembly
A smaIl, loose pie ce of non-reactive material, such as not
consists of a set of cylindrical, ftat-bottomed glass vessels;
more than a few turns of wire helix, may be attached to
a set of glass reciprocating cylinders; inert fittings (stainless
dosage units that would otherwise ftoat. An alternative sinker
steel type 316 or other suitable material) and screens that are
device is shown in Figure 2.9.3.-3. Other validated sinker
made of suitable nonsorbing and nonreactive material, and
devices may be used.
that are designed to fit the tops and bottoms of the
reciprocating cylinders; a motor and drive assembly to
reciprocate the cylinders verticaIly inside the vessels, and if
desired, index the reciprocating cylinders horizontaIly to a
different row of vessels. The ves seIs are partiaIly immersed in
a suitable water-bath of any convenient size that permits
holding the temperature at 37 ± 0.5 oC during the test.
1 The marerials musr nor sorb, reaCL, or i"!elfere wirh rhe preparaLion 10 be
No part of the assembly, incIuding the environment in which
resud. the assembly is placed, contributes significant motion,
2 JI a caVe!" is used, ir provides sufficiem openings 10 a//ow ready insertion 01
agitation, or vibration beyond that due to the smooth,
rhe rhennomerer and wirhdrawa/ 01 samples.
9.4 to 10.1
L()
o
+1
o
m
+1 74.5 ± 0.5
o
-.i
A and B dimensions do not vary more than 0.5 mm when part is rotated on center line axis. Tolerances
are ± 1.0 mm unless otherwise stated.
Figure 2.9.3.-2 . - Apparatus 2, Paddle stirring element
vertically reciprocating cylinder. A device is used that allows cylinders is preferable. The ves seis are provided with an
the reciprocation rate to be selected and mainrained at the evaporaríon cap that remains in place for the duraríon of the
specified dip rate, within ± 5 per cenr. An apparatus that test. The componenrs conform to the dimensions shown in
permits observation of the prepararíons and reciprocating Figure 2.9.3 .-4 unless otherwise specified.
3.5-4.0 3.0-3.5 3.5-4.0

A B
3.5-4.0 3.5-4.0
25-26
.1 12.0 ± 0.2
A: acid-resistant wire c1asp B: acid-resistant wire support

Figure 2.9.3.-3. - Alternative sinker
Apparatus 4 (Flow-through cell) The assembly consists PROCEDURE

of a reservoir and a pump for the dissolution medium; Apparatus 1 and 2
a flow-through cell; a water-bath that maintains the
Conventional-release solid dosage forros
dissolution medium at 37 ± 0.5 oc. Use the specified cell
size. Procedure Place the stated volume of the dissolution
mediurn (± 1 per cent) in the vessel of the specified
The pump forces the dissolution medium upwards through
apparatus. Assemble the apparatus, equilibrate the dissolution
the flow-through cel!. The pump has a delivery range
medium to 37 ± 0.5 oC, and remove the thermometer.
between 240 mlJh and 960 mlJh, with standard flow rates
The test may also be carried out with the thermometer in
of 4 mlJmin, 8 mlJmin, and 16 mlJmin. It must deliver a
place, provided it is shown that results equivalent to those
constant flow (± 5 per cent of the nominal flow rate);
obtained without the thermometer are obtained.
the flow profile is sinusoidal with a pulsation of 120 ± 10
pulses/min. A pump without pulsation may also be used. Place 1 dosage unit in the apparatus, taking care to exclude
Dissolution test procedures using the flow-through cell must air bubbles from the surface of the dosage unit. Operate the
be characterised with respect to rate and any pulsation. apparatus at the specified rate. Within the time interval
specified, or at each of the times stated, withdraw a spe~imen
The flow-through cell (se e Figures 2.9.3.-5 and 2.9.3.-6) of
from a zone midway between the surface of the dlSSolutIon
transparent and inert material is mounted vertically, with a
medium and the top of the rotating basket or blade, not less
filter system that prevents escape of undissolved particles
than 1 cm from the vessel wal!. Where multiple sampling
from the top of the cell; standard cell diameters are 12 mm
times are specified, replace the aliquots withdrawn for
and 22.6 mm; the bottom cone is usually filled with small
analysis with equal volumes of fresh dissolution medium at
glass beads of about 1 mm diameter, with 1 bead of about
37 oC or, where it can be shown that replacement of the
5 mm positioned at the apex to protect the fluid entry tube;
medium is not necessary, correct for the volume change in
a tablet holder (see Figures 2.9.3.-5 and 2.9.3.-6) is available
the calculation. Keep the vessel covered for the duration of
for positioning of special dosage forms. The cell is immersed
the test and verify the temperature of the medium at suitable
in a water-bath, and the temperature is maintained at
times. Perform the analysis using a suitable assay method3 •
37 ± 0.5 oc.
Repeat the test with additional dosage units.
The apparatus uses a clamp mechanism and 2 O-rings for
If automated equipment is used for sampling or the
the fixation of the cell assembly. The pump is separated from
apparatus is otherwise modified, verification that the
the dissolution unit in order to shield the latter against any
modified apparatus will produce results equivalent to those
vibrations originating from the pump. The position of the
obtained with the apparatus described in this chapter, is
pump must not be on a level higher than the reservoir flasks.
necessary.
Tube connections are as short as possible. Use suitably inert
tubing, su eh as polytetrafluoroethylene, with a 1.6 mm inner Dissolution medium A suitable dissolution medium is
diameter and inert flanged-end connections. used. The volume specified refers to measurements made
between 20 oC and 25 oC. If the dissolution medium is a
Apparatus suitability The determination of suitability of
buffered solution, adjust the solution so that its pR is within
the apparatus to perform dissolution testing must include
0.05 units of the specified pR. Dissolved gases can cause
conformance to the dimensions and tolerances of the
bubbles to form, which may change the results of the test.
apparatus as given aboye. In addition, critical test parameters
In such cases, dissolved gases must be removed prior to
that have to be monitored periodically during use include
testing4 .
volume and temperature of the dissolution medium, rotation
speed (Apparatus 1 and 2), dip rate (Apparatus 3), and flow
3 Test specimens are filtered immediately upon sampling unless filtration is
rate of medium (Apparatus 4).
demonstrated 10 be unnecessary. Use an inert filter that does not cause
Determine the ~cceptable performance of the dissolution test adsorption of the active substance or contain extractable substances that
assembly periodically. would inteifere with the analysis
4 A method of deaeration is as follows: heat the medium, while stirring
gently, 10 about 41 oC, immediately filter under vacuum using a filter having
a porosity of 0.45 11m or less, with vigorous stirring, and continue. sttmng
under vacuum for about 5 mino Other validated deaeration techmques for
removal of dissolved gases may be used.
Time Where a single time specification is given, the test " 50.8 ± 1 _,
may be conc1uded in a shorter period if the requirement for 38 .1 ± 1 I
minimum amount dissolved is meto Samples are to be I" ..
Air holes
~f2J3.9±0.1
withdrawn only at the stated times, within a tolerance of
± 2 per cent. r----rf,1-+-I-1,1-'-T--..,
'
Prolonged-release solid dosage forms l
Procedure Proceed as described for conventional-release I
I
dosage forms.
Dissolution medium Proceed as described for
<Xl
+1
(O
(O
I
Evaporation cap
conventional-release dosage forms.

I
Time The test-time points, gene rally 3, are expressed in
hours .
Delayed-release solid dosage forms
Procedure Use Method A or Method B.
Method A
- Acid stage Place 750 mL of 0.1 M hydrochloric acid in
the vessel, and assemble the apparatus. Allow the medium
¡Ii~---- Mesh screen
to equilibrate to a temperature of 37 ± 0.5 oC. Place
1 dosage unit in the apparatus, cover the vessel and
operare the apparatus at the specified rate. After 2 h of
operation in 0.1 M hydrochloric acid, withdraw an aliquot
of the fluid and proceed immediately as directed under
Buffer stage. Perform an analysis of the aliquot using a 1
1
suitable assay method. 1
1
- Buffer stage Complete the operations of adding the +1 1
o 11_">----- Glass reciprocating
buffer and adjusting the pH within 5 min. With the o : cylinder
apparatus operating at the rate specified, add to the fluid
1
in the vessel 250 mL of a 0.20 M solution of trisodium 1
phosphate dodecahydrate R that has been equilibrated to 1
1
37 ± 0.5 oC. Adjust, if necessary, with 2 M hydrochloric 1
1
1
acid R or 2 M sodium hydroxide R to a pH of 6.8 ± 0.05.
Continue to operate the apparatus for 45 min, or for the
specified time. At the end of the time period, withdraw an
j-~<Xl~[t::~'III~1
T-.1 _"e__-- Mesh screen
aliquot of the fluid and perform the analysis using a
suitable assay method. 1
1 I
Method B 1
1 47 ± 1.4
- Acid Stage Place 1000 mL of O. 1 M hydrochloric acid in 1" "1
1
the vessel and assemble the apparatus. Allow the mediurn
to equilibrate to a temperature of 37 ± 0.5 oC. Place
1 dosage unit in the apparatus, cover the vessel, and j I
operate the apparatus at the specified rate. After 2 h of : 1
operation in 0.1 M hydrochloric acid, withdraw an aliquot

of the fluid, and proceed immediately as directed under ____ Glass vessel
Buffer stage. Perform an analysis of the aliquot using a i
1
I
suitable assay method. 1 1
<Xl
+1
- Buffer stage ' For this stage of the procedure use buffer o
that has previously been equilibrated to a temperature of
37 ± 0.5 oC. Drain the acid fram the vessel and add
i
1
I
1 1
1000 mL of pH 6.8 phosphate buffer, prepared by mixing
3 volurnes of 0.1 M hydrochloric acid with 1 volurne of a
0.20 M solution of trisodium phosphate dodecahydrate R
and adjusting, if necessary, with 2 M hydrochloric acid R
i
1
I
1 1
or 2 M sodium hydroxide R to a pH of 6.8 ± 0.05. This
may also be accomplished by removing fram the
apparatus the vessel containing the acid and replacing it i I :
with another vessel, containing the buffer and transferring
the dosage unit to the vessel containing the buffer.
:
L ___ + ___ -l
1 :
Continue to operate the apparatus for 45 min, or for the

specified time. At the end of the time period, withdraw an I
aliquot of the fluid and perform the analysis using a
suitable assay method. Figure 2.9.3.-4. - Apparatus 3, glass vessel and reciprocating
Time AH test times stated are to be observed within a cylinder
tolerance of ± 2 per cent, unless otherwise specified.
Dimensions in millimetres unless otherwise specified
2014 Appendix XII B V -A341
f**",**"f*B*B _____ Filler chamber

Sieve
l
- - - - - d = 0.2 w = 0.45
020 0.2
L!)
o 022.6 ± 0.2
+1
L!)
Lli
C') -- ___ Score for lhe holder
L!)
C')
¿
'E
I
-,- 00.8 ± 0.05
L!)
<D
L!)
o
L!)
N
o
+1
L!)
24 .0 + 8. 5
N
Figure 2.9.3.-5. - Apparatus 4, large cell for tablets and capsules (top), tablet holder for the large cell (bottom)
Dimensions in millimetres unless otherwise specified
V -A342 Appendix XII B 2014
l()
c:i
-tl
l()
l()
1--lf.*,)f.-4¡'-lf-~H+i?--l<L.:jj ____ Filler chamber
tt:;ltj~jS:4Ztt:Zt;~~ ----Sieve d = 0.2 w = 0.45

020 ± 0.2
l() 012 ± 0.2

c:i
-tl
o
l()
_..
_ -- - Score tor Ihe holder
~~------~
Ei--- -==-l::iJW.__- - - 00.8 ± 0.05
- - : - - f - - I I - - - - - - - 03
l()
<O
<O
~
+ 0.5
13.5 O
Figure 2.9.3.-6. - Apparalus 4, smal! cel! for lablels and capsules (lop), lablel holder for lhe smal! cel! (bottom)
Dimensions in millimelres unless olherwise specified
Apparatus3 cylinder moves through a total distance of 9.9-10.1 cm.

Conventional-release solid dosage forms Within the time interval specified, or at each of the times
Procedure Place the stated volume of the dissolution stated, raise the reciprocating cylinders and withdraw a
medium ( ± 1 per cent) in each vessel of the apparatus. portion of the medium from a zone midway between the
Assemble the apparatus, equilibrate the dissolution medium surface of the dissolution medium and the bottom of each
to 37 ± 0.5 oC, and remove the thermometer. Place vessel. Perform the analysis as directed. If necessary, repeat
1 dosage unit in each of the reciprocating cylinders, taking the test with additional dosage units .
care to exclude air bubbles from the surface of each dosage Replace the aliquot withdrawn for analysis with equal
unit, and irnmediately operate the apparatus as specified. volumes of fresh dissolution medium at 37 oC or, where it
During the upward and downward stroke, the reciprocating can be shown that replacement of the medium is not
necessary, correct for the volume change in the calculation. Table are percentages of the labelled content so that these
Keep the vessel covered with the evaporation cap for the values and Q are in the same terms,
duration of the test and verify the temperature of the
medium at suitable times. Table 2.9,3.-1
Dissolution medium Proceed as described for Level Number Acceptance criteria
tested
conventional-release dosage forms under Apparatus 1 and 2.
51 6 Each unit is not less than Q + 5 per cent.
Time Proceed as described for conventional-release dosage
forms under Apparatus 1 and 2. 5, 6 Average of 12 units (51 + 5, ) is equal to or greater than Q,
and no unit is less than Q - 15 per cent.
Prolonged-re1ease dosage forms 53 12 Average of 24 units (51 + 52 + 53) is equal to or greater
Procedure Proceed as described for conventional-release than Q, not more than 2 units are less lhan Q - 15 per cent,
and no is less than Q - 25 per cent
dosage forms under Apparatus 3.
Dissolution medium Proceed as described for prolonged- Prolonged-release dosage forms
release dosage forms under Apparatus 1 and 2.
Unless otherwise specified, the requirements are met if the
Time Proceed as described for prolonged-release dosage quantities of active substance dissolved from the dosage units
forms under Apparatus 1 and 2. tested conform to Table 2.9.3.-2. Continue testing through
Delayed-release dosage forms the 3 levels unless the results conform at either LI or L 2 ,
Procedure Proceed as described for delayed-release dosage Limits on the amounts of active substance dissolved are
forms, Method B, under Apparatus 1 and 2, using one row expressed in terms of the percentage of labelJed contento
of ves seIs for the acid stage media and the following row of The limits embrace each value of Q" the amount dissolved at
ves seIs for the buffer stage media, and using the volume of each specified fractional dosing interva!. Where more than
medium specified (usually 300 mL) . one range is specified, the acceptance criteria apply
Time Proceed as directed for delayed-release dosage forms individually to each range,
under Apparatus 1 and 2.
Table 2,9,3,-2
Apparatus 4 Level Number Acceptance criteria
Conventional-release dosage forms tested
LI 6 No individual value lies outside each of the stated ranges
Procedure Place the glass beads into the cell specified. and no individual value is less than the stated amount at
Place 1 dosage unit on top of the beads or, if specified, on a the final test time,
wire carrier. Assemble the filter head and fix the parts L, 6 The average value of the 12 units (L I + L,) lies within each
together by means of a suitable c1amping device. Introduce of the stated ranges and is not less than the stated amount
at the final test time ; non e is more than 10 per cent of
by the pump the dissolution medium warmed to labelled content outside each of the stated ranges; and none
37 ± 0.5 oC through the bottom of the cell to 'obtain the is more than 10 per cent oflabelled content below the stated
amount at the final test time,
fiow rate specified and measured with an accuracy of
L, 12 The average value of the 24 units (L I + L, + L,) lies within
5 per cent. Collect the eluate by fractions at each of the each of the stated ranges, and is not less than the stated
times stated. Perform the analysis as directed, Repeat the test amount at the final test time; not more than 2 of the 24 units
with additional dosage units , are more than 10 per cent of labelled content outside each
of the stated ranges; not more than 2 of the 24 units are
Dissolution medium Proceed as described for more than 10 per cent of labelled content below the stated
conventional-release dosage forms under Apparatus 1 and 2. amount at the final test time; and none of the units is more
than 20 per cenl of labelled content outside each of the
Time Proceed as described for conventional-release dosage stated ranges or more than 20 per cent of labelled content
forms under Apparatus 1 and 2, below the stated amount at the final test time,
Prolonged-release dosage forms

Delayed-release dosage forms
Procedure Proceed as described for conventional-release
Acid stage Unless otherwise specified, the requirements of
dosage forms under Apparatus 4,
this portion of the test are met if the quantities, based on the
Dissolution medium Proceed as described for percentage of the labelled content of active substance
conventional-release dosage forms under Apparatus 4 , dissolved from the units tested conform to Table 2.9,3 ,-3 ,
Time Proceed as described for conventional-release dosage Continue testing through the 3 levels unless the results of
forms under Apparatus 4, both acid and buffer stages conform at' an earlier leve!.
De1ayed-release dosage forms
Table 2.9.3.-3
Procedure Proceed as described for delayed-release dosage
forms under Apparatus 1 and 2, using the specified media. Level Number Acceptance criteria
tested
Time Proceed as described for delayed-release dosage Al 6 No individual value exceeds 10 per cent dissolved,
forms under Apparatus 1 and 2,
A, 6 The average value of the 12 units (A l + A,) is not more than
10 per cent dissolved, and no individual unit is greater than
INTERPRETAnON 25 per cent dissolved,
Conventional-release solid dosage forms A, 12 The average value of the 24 units (Al + A, + A3 ) is not more
than 10 per cent dissolved, and no individual unit is greater
Unless otherwise specified, the requirements are met if the than 25 per cent dissolved,
quantities of active substance dissolved from the dosage units
tested conform to Table 2.9,3,-1. Continue testing through Buffer stage Unless otherwise specified, the requirements
the 3 levels unless the results conform at either SI or S2' are met if the quantities of active substance dissolved from
The quantity Q, is the specified amount of dissolved active the units tested conform to Table 2.9.3.-4. Continue testing
substance, expressed as a percentage of the labelled contenti through the 3 levels unless the results of both stages conform
the 5 per cent, 15 per cent, and 25 per cent values in the at an earlier leve!. The value of Q in Table 2.9 .3.-4 is
75 per cent dissolved unless otherwise specified.
The quantity, Q, is the specified total amount of active In the determination of the dissolution rate of the active
substance dissolved in both the acid and buffer stages, substance(s) of a solid dosage form, the following are to be
expressed as a percentage of the labelled contento specified:
The S per cent, 15 per cent and 25 per cent values in the - the apparatus to be used, and in cases where the flow-
Table are percentages of the labelled content so that these through apparatus is specified, which flow-through cell is
values and Q are in the same terms. 10 be used;
- the composition, the volume and the temperature of the
Table 2.9.3.-4 dissolution medium;
Level Number Acceptance criteria
tested - the rotation speed or the flow rate of the dissolution
B, 6 No unit is less than Q + 5 per cenl medium;
- the time, the method and the amount for sampling of the
B, 6 The average value of the 12 units (B, + B,) is equal to or
greater than Q, and no unit is less than Q -15 per cenl. test solution or the conditions for continuous monitoring;
B3 12 The average value of the 24 units (B, + B, + Bol is equal - the method of analysis;
to or greater Ihan Q. not more than 2 units are less than
Q - 15 per cen\, and no unit is less than Q - 25 per cenl. - the acceptance criteria.
The choice of apparatus to be used depends on the physico-
Recommendations 01'1 dissolution testing are given in general chemical characteristics of the dosage formo When a large
chapter 5.17.1. quantity of dissolution medium is required to ensure sink
conditions, or when a change of pH is necessary, the flow-
Monographs 01 the British Pharmacopoeia through apparatus may be preferred.
The following additional points apply to monographs of the
British Pharmacopoeia. EXPERIMENTAL TESTING CONDITIONS
APPARATUS The use of the basket and the paddle apparatus and the
The choice of the apparatus to be used depends on the reciprocating cylinder apparatus is generally based on the
physico-chemical characteristics of the dosage formo When principie of operating under sink conditions, i.e . in such a
this Appendix is invoked in an individual tablet or capsule manner that the material already in solution does not exert a
monograph of the British Pharmacopoeia, use Apparatus I significant modifying effect on the rate of dissolution of the
unless otherwise directed. remainder. Sink conditions normally occur in a volume of
PROCEDURE dissolution medium that is at least 3-10 times the saturation
The dissolution medium is that specified in the individual volume.
monograph. Unless otherwise indicated in the monograph, In general, an aqueous medium is used. The composition of
withdraw samples at 45 minutes . the medium is chosen on the basis of the physico-chemical
Where one tablet or capsule is directed to be placed in the characteristics of the active substance(s) and excipientes)
apparatus, for each of the six tablets or capsules tested the within the range of conditions to which the dosage form is
amount of active ingredient in solution is not less than 70% likely to be exposed after its administration. This applies in
of the prescribed or stated amount, unless otherwise specified particular to the pH and the ionic strength ofthe dissolution
in the monograph, except that if one fails this requirement a medium.
further six may be tested individualIy and alI must comply. The pH of the dissolution medium is usually set between
Where two or more tablets or capsules are directed to be pH 1 and pH 8. In justified cases, a higher pH may be
placed together in the apparatus, a total of six replicate tests needed. For the lower pH values in the acidic range, 0.1 M
are carried out. In each test the amount of active ingredient hydrachloric acid is normally used. Recommended dissolution
in solution per tablet or capsule is not less than 70% of the media are described hereafter.
prescribed or stated amount, unless otherwise specified in the Water is recommended as a dissolution medium only when it
monograph. No retesting is permitted. is proven that the pH variations do not have an influence on
Where capsule shells interfere with the analysis, remove the the dissolution characteristics.
contents of no fewer than six capsules as completely as In specific cases, and subject to approval. by the competent
possible and dissolve the empty capsule shells in the specified authority, dissolution media may contain enzymes,
volume of dissolution medium. Carry out the test as directed surfactants, further inorganic substances and organic
in the individual monograph and make any necessary substances. For the testing of preparations containing poorly
correction. Correction factors should not be greater than aqueous-soluble active substances, modification of the
25% of the labelled contento medium may be necessary. In such circumstances, a low
concentration of surfactant is recommended; it is
recommended to avoid the use of organic solvents.
Gases dissolved in the dissolution medium can affect the
ANNEX results of the dissolution test. This is true in particular for the
Recornrnendations on Dissolution flow-through apparatus, where de-aeration of the medium is
necessary to avoid the formation of gas bubbles in the flow-
Testing through cell. A suitable method of de-aeration is as follows:
(Ph. Eur. general text 5. 17. 1) heat the medium while stirring gently to about 41 °C,
This general chapter is non-mandatoryj it provides information on immediately filter under vacuum using a filter with a porosity
dissalution testing, on recommended dissolutian media and 01'1 the of 0.45 11m or less, with vigorous stirring, and continue
expression of dissolution specifications for oral dasage farms (see stirring under vacuum for about S mino Other de-aeration
general chapter 2. 9.3. Dissolution test far solid dosage forms). This techniques for removal of dissolved gases may be used.
informatian represents generally accepted parameters used in the Using the paddle or basket apparatus, the volume of
field of dissolution. dissolution medium is normally 500-1000 mL. A stirring
speed of between 50 rlmin and 100 r/min is normally chosen; - Acetate buffer solution pH 5.8. Dissolve 6.23 g of sodium
it must not exceed 150 r/min. acetate R in water R. Add 2.1 mL of 2 M ace/ic acid and
For the f1ow-through appararus, the liquid f10w rate is dilute to 1000.0 mL with water R .
normally set between 4 mUmin and 50 mUrnin. Phosphate buffer solutions
For preparing buffers with the pH values indicated in
RECOMMENDED DISSOLUTION MEDIA
Table 5.17.1.-3, mix 250.0 mL of 0. 2 M potassium dihydrogen
The following dissolution media may be used. phosphate R and the specified volume of 0.2 M sodium
hydroxide, and dilute to 1000.0 mL with water R.
Table 5.17.1.-1. - Examples of dissolution media
pH Dissolution media Table 5.17.1.-3. - Phosphate buffer solutions
pH 1.0 HCI pH 5.8 6.0 6.2 6.4 6.6 6.8
NaOH
pH 1.2 NaCI. HCI (mL) 18.0 28.0 40.5 58.0 82.0 112.0
pH 1.5 NaCI. HCI pH 7.0 7.2 7.4 7.6 7.8 8.0

pH 4.5 Phosphate or acetate buffer NaOH 145.5 173.5
(mL) 195.5 212.0 222.5 230.5
pH 5.5 and pH 5.8 Phosphate or aceta te buffer
pH 6.8 Phosphate buffer
Other phosphate buffer solutions
pH 7.2 and pH 7.5 Phosphate buffer
- Phosphate buffer solution pH 4.5. Dissolve 13.61 g of
potassium dihydrogen phosphate R in 750 mL of water R.
The composition and preparation of these various media are Adjust the pH if necessary with 0.1 M sodium hydroxide or
indicated below. with 0.1 M hydrochloric acid. Dilute to 1000.0 mL with
water R .
Hydroehlorie aeid media
- Phosphate buffer solution pH 5.5 R.
- 0.2 M hydrochlon'c acid.
- Phosphate buffer solution pH 6.8 Rl.
- 0.2 M sodium ehlonde. Dissolve 11.69 g of sodium
ehloride R in water R and dilute to 1000.0 mL with the - Buffer solution pH 7.2 R.
same solvento - 0.33 M phosphate buffer solution pH 7.5 R.
For preparing media with the pH values indicated in Simulated intestinal fluid pH 6.8
Table 5.17.1.-2, mix 250.0 mL of 0.2 M sodium ehloride and Mix 77.0 mL of 0.2 M sodium hydroxide, 250.0 mL of a
the specified volume of 0.2 M hydrochloric acid, and dilute to solution containing 6.8 g of potassium dihydrogen phosphate R,
1000.0 mL with water R. and 500 mL of water R . Add 10.0 g of pancreas powder R,
mix and adjust the pH ifnecessary. Dilute to 1000.0 mL
Table 5.17.1.-2. - Hydrochloric acid media with water R.
pH HCl(mL) Simulated gas trie fluid
1.2 425.0 Dissolve 2.0 g of sodium chloride R and 3.2 g of pepsin
powder R in water R, add 80 mL of 1 M hydrochloric acid and
1.3 336.0
dilute to 1000.0 mL with water R. If required, pepsin powder
1.4 266.0 may be omitted.
1.5 207.0 Inereasing pH
1.6 162.0 For a test involving increasing pH, one of the following
1.7 130.0 sequences may be used:
1.8 102.0
1.9 81.0
Time (h) 0-1 1-2 I 2 -3 I 3 -4 I 4-5 I 5-6 16 - 7 I 7
pH 1.0
2.0 65.0
pH 1.2 6.8
2.1 51.0
2.2 39.0
pH 1.2 2.5 I 4.5
I 7.0
I 7.5
pH 1.5 4.5 7.2
I
The hydrochloric acid media may also be prepared by
replacing sodium chloride with potassium chloride. To achieve this pH variation, it is possible either:
Acetate buffer solutions - to substitute one buffer solution for another (whole
- 2 M aeetic acid. Dilute 120.0 g of glacial aeetie acid R to substirution) ;
1000.0 mL with water R. - to remove only half of the medium each time (half change
- Aeetate buffer solution pH 4.5. Dissolve 2.99 g of sodium method) and replace it with a buffer solution of higher
aeetate R in water R. Add 14.0 mL of 2 M acetic acid and
pH: the initial pH is 1.2 and the second solution is
dilute to 1000.0 mL with water R. phosphate buffer solution pH 7.5; or,
- Acetate buffer solution pH 5.5. Dissolve 5.98 g of sodium - to an inirial solution at pH 1.5, to add adose of a powder
acetate R in water R . Add 3.0 mL of 2 M acetic acid and
mixture containing tris(hydroxymethyl)aminomethane R and
dilute to 1000.0 mL with water R . anhydrous sodium acetate R to obtain pH 4 .5 and a second
dose to obtain pH 7.2, as described below:
- hydrochloric acid pH 1.5: dissolve 2 g of sodium unintended rapid release of the active substance ('dose
chloride R in water R, add 31. 6 mL of 1 M hydrochlonc dumping'). It is therefore set after a testing period
acid and dilute to 1000.0 mL with water R; corresponding to a dissolved amount typically of 20 per cent
- buffer solution pH 4.5: mix 2.28 g of to 30 per cent. The 2nd specification point defines the
tns(hydroxymethyl)aminomethane R with 1.77 g of dissolution panern and so is set at around 50 per cent
anhydrous sodium acetate R; dissolve this mixture in the release. The final specification point is intended to ensure
hydrochloric acid pH 1.5 described aboye; almost complete release, which is generally understood as
more than 80 per cent release ,
- buffer solution pH 7.2: mix 2.28 g of
tris(hydroxymethyl)aminomethane R with 1.77 g of Delayed-release dosage forms
anhydrous sodium acetate R; dissolve this mixture in the A delayed-release dosage form may releas e the active
buffer solution pH 4.5 described aboye. substance(s) fractionally or totalIy according to the
The ftow-through cell may be used for the continuous formulation design when tested in different dissolution
change of pH. media, e.g. in increasing pH conditions. Dissolution
specifications therefore have to be decided on a case-by-case
QUALIFlCATlON AND VALIDATlON basis .
Due to the nature of the test method, quality by design is an Gastro-resistant do sage forms require at least 2 specification
important qualification asp ect for in vitro dissolution test points in a sequential test and 2 different specifications in a
equipment. Any irregularities such as vibration or undesired paralle! test. In a sequential test, the 1st specification point
agitation by mechanical imperfections are to be avoided. represents an upper limit and is set after 1 h or 2 h in acidic
Qualification of the dissolution test equipment has to medium, and the 2nd after a pre-set time period of testing in
consider the dimensions and -tolerances of the apparatus. an adequate buffer solution (preferably pH 6.8).
Critical test parameters, such as temperature and volume of In most cases the acceptance criteria at level Bl are that at
dissolution medium, rotation speed or liquid ftow rate, least 80 per cent of the active substance is released. This
sampling probes and procedures, have to be monitored corresponds to a Q value of 75 per cent, since, as referred to
periodically during the periods of use. in Table 2.9.3.-4, for level Bl the individual value of each of
The performance of the dissolution test equipment may be the 6 units tested is not less than Q + 5 per cent, i,e . not less
monitored by testing a reference product that is sensitive to than 80 per cent.
hydrodynamic conditions. Such tests may be performed
periodically or continu{lUsly for comparative reasons with 2. Dissolution Test for Transdermal Patches
other laboratories . (Ph . Eur. method 2.9.4)
During testing, critical inspection and observation are This test is used to determine the dissolution rate of the
required. This approach is especially important to explain active ingredients of transdermal patches.
any outlying results. 1. Disk assembly method
Validation of automated systems, whether concerning the Equipment Use the paddle and vessel assembly from the
sampling and analyrical part or the dissolution media paddle apparatus described in the dissolution test for solid
preparation and test performance, has to consider accuracy, oral dosage forms (2.9.3) with the addition of a stainless
precision, and the avoidance of contamination by any steel disk assembly (SSDA) in the form of a net with an
dilutions, transfers, c1eaning and sample or solvent aperture of 125 ¡.lm (see Figure 2.9 .4.-1) .
preparation procedures.
EXPRESS ION OF DISSOLUTlON 125 1.1 m mesh

stainless steel net 4.5 mm
SPECIFlCATlONS FOR ORAL DOSAGE FORMS
1- 1
The dissolution specification is expressed in terms of the
quantity (Q) of active substance dissolved in a specified time,
expressed as a percentage of the content stated on the \J D 13.3mm
product labe!'
Conventional-release dosage forms
In most cases, when tested under reasonable and justified test
conditions, the acceptance criteria at level SI are that at least
80 per cent of the active substance is re!eased within a
specified time, typically 45 min or less. This corre's ponds to a
Q value of 75 per cent, since, as referred to in
Table 2.9 .3.-1 , for leve! SI the individual value ofeach ofthe
6 units tested is not less than Q + 5 per cent, i.e. not les s
than 80 per cent.
Typically, a single-point acceptance criterion is sufficient to
demonstrate that the majority of the active substance has
been released,although in certain circumstances it may be
necessary to test at additional time point(s), in order to
demonstrate adequate dissolution.
Prolonged-release dosage forms ~ 41 .2mm ~
The dissolution test acceptance criteria for prolonged-release
dosage forms is riormally expected to consist of 3 or more Figure 2.9.4.-1. - Disk assembly
points. The 1s t specification point is intended to prevent
The SSDA holds the system at the bottom of the vessel and adversely affect the physico-chemical properties of the patch
is designed to minimise any dead volume between the SSDA (see Figure 2.9.4.-3).
and the bottom of the vessel. The SSDA holds the patch fiat,
with the release surface uppermost and parallel to the bottom 20 mm
of the paddle blade. A distance of 25 ± 2 mm between the @:;¡
32mm ~
bottom of the paddle blade and the surface of the SSDA is 1-_ _ _ _..... 45 mm
maintained during the test (see Figure 2.9.4 .-2) . ~ 50mm ~ _Nuls
The temperature is maintained at 32 ± 0.5 oc. The vessel
may be covered during the test to minimise evaporation.
o
o k._ _~50~m~m~_-+)
19.63 cm'
~ Cover
e
o
==::===:::::~_ ____ '- 8.5 mm
Dissolulion vessel
:~_M,m~"
- t - - - - t - - Paddle
2.6 mm ""'===~b~~==~:::::::::::::;:!tl-_-_-_-_-_--= BOIIS

1 Rubber ring
/ -, t ¡ - - --+ Reservoir
~___-===-+_-I--_--'- 9.6 mm
52mm
A
Disk assembly 21.23 cm'
Figure 2.9.4.-2. - Paddle and disk 70.5 mm
27mm
Procedure Place the prescribed volume of the dissolution 1 - -_ 38mm
medium in the vessel and equilibrate the medium to the 1 - - -- -+45 mm
prescribed temperature. Apply the patch to the SSDA, 1-------~ 52mm
ensuring that the release surface of the patch is as fiat as
possible. The patch may be attached to the SSDA by a
prescribed adhesive or by a strip of a double-sided adhesive
tape. The adhesive or tape are previously tested for the Figure 2.9.4.-3. - Extraction cel!
absence of interference with the assay and of adsorption of
the active ingredientes). Press the patch, release surface Support The central part of the support forms a cavity
facing up, onto the side of the SSDA made adhesive. intended to hold the patch. The cavity has a depth of
The applied patch must not overlap the borders of the 2.6 mm and a diameter that is appropriate to the size of the
SSDA. For this purpose and provided that the preparation is patch to be examined. The following diameters can be used:
homogeneous and uniformly spread on the outer covering, 27 mm, 38 mm, 45 mm, 52 mm, corresponding to volumes
an appropriate and exactly measured piece of the patch may of 1.48 mL, 2.94 mL, 4.13 mL, 5.52 mL, respectively.
be cut and used for testing the dissolution rateo This
Cover The cover has a central opening with a diameter
procedure may also be necessary to achieve appropriate sink
selected according to the size of the patch to be examined.
conditions. This procedure must not be applied to
The patch can thus be precisely centred, and its releasing
membrane-type patches . Place the patch mounted on the
surface limited. The following diameters may be used:
SSDA fiat at the bottom of the vessel with the release
20 mm, 32 mm, 40 mm, 50 mm corresponding to areas of
surface facing upwards. Irnmediately rota te the paddle at
3.14 cm2 , 8.03 cm 2 , 12.56 cm 2 , 19.63 cm2 , respectively.
100 r /min, for example. At predetermined intervals,
withdraw a sample from the zone midway between the The cover is held in place by nuts screwed onto bolts
projecting from the support. The cover is sealed to the
surface of the dissolution medium and the top of the blade,
support by a rubber ring set on the reservoir.
not less than 1 cm from the vessel wall.
Extractíon cell The cell holds the patch fiat, with the releas e
Perform the assay on each sample, correcting for any volume
surface uppermost and parallel to the bottom of the paddle
losses, as necessary. Repeat the test with additional patches.
blade. A distance of 25 ± 2 mm is maintained between the
2. Cell method paddle blade and the surface of the patch (se e Figure
Equipment Use the paddle and vessel assembly from the 2.9.4.-4). The temperature is maintained at 32 ± 0.5 oc.
paddle apparatus described in the dissolution test for solid The vessel may be covered during the test to minimise
oral dosage forms (2.9.3) with the addition of the extraction evaporation.
cell (cel!) . Procedure Place the prescribed volume of the dissolution
The cell is made of chemically inert materials and consists of medium in the vessel and equilibrate the medium to the
a support, a cover and, if necessary, a membrane placed on the prescribed temperature. Precisely centre the patch in the cel!
patch to iso late it from the medium that may modify or with the releasing surface uppermost. Close the cell, if
necessary applying a hydrophobic substance (for example,
t
1.270
+
Four holes at 1.111 dia.
equally spaced on 2.540
Interference tit
dia. b.c. at 63.4 ± OS
angle to surface
1. ,12 40.640 ]
Maximum radius 0.300 -~~::::::~,;:~:;:tri3 . 967
-t- j 5.079
:+2.14611 t
¡+2. 134 1 -~--,-
I r This adaptor
TOLERANCES : ± 0.0127
I
f..2.045
3TO section is to
be used tor
larger systems
FINISH : Degrease befo re
final assembly or rod
and cylinder
- ,- 9.383
MATERIAL: Stainless steel -!:::::j:::::::::;~ 5 .~ 12 1
Figure 2.9.4.-5. - Cylinder stirring element

Dimensions in centimetres
petrolatum) to the fiat surfaces to ensure the seal, and ensure Perform the assay on each sample, correcting for any volume
that the patch stays in place. Introduce the cell fiat into the losses, as necessary. Repeat the test with additional patches.
bottom of the vessel with the cover facing upwards. 3. Rotating cylinder method
Irnmediately rotate the paddle, at 100 r/min for example.
At predetermined intervals, withdraw a sample from the zone Equipment Use the assembly of the paddle apparatus
midway between the surface of the dissolution medium and described in the dissolution test for solid oral dosage forms
the top of the paddle blade, not less than 1 cm from the (2.9.3). Replace the paddle and shaft with a stainless steel
vessel wall. cylinder stirring element (cylinder) (se e Figure 2.9.4.-5).
The patch is placed on the cylinder at the beginning of each
test. The distance between the inside bottom of the vessel
and the cylinder is maintained at 25 ± 2 mm during the test.
The temperature is maintained at 32 ± 0.5 oc. The vessel is
'-- ,.-'
covered during the test to minimise evaporation,
Procedure Place the prescribed volume of the dissolution
medium in the vessel and equilibrate the medium to the
Paddle
prescribed temperature. Remove the protective liner from the
patch and place the adhesive side on a piece of suitable inert
porous membrane that is at least 1 cm larger on all sides
Dissolution vessel than the patch. Place the patch on a clean surface with the
membrane in contact with this surface. Two systems for
adhesion to the cylinder may be used:
- apply a suitable adhesive to the exposed membrane
\ J borders and, if necessary, to the back of the patch,
..., :: ,.
/ 1 2
5±2 mm - apply a double-sided adhesive tape to the external wall of
the cylinder.
.... _-.- ....... --------- ,. / Extraction cell Using gentle pressure, carefully apply the non-adhesive side
~ /' of the patch to the cylinder, so that the release surface is in
contact with the dissolution medium and the long axis of the
Figure 2.9.4.·4. - Paddle over extraction cell patch fits around the circumference of the cylinder.
The system for adhesion used is previously tested for absence beginning of the test, chamber A requires air removal via a
of interference with the assay and of adsorption of the active small orifice connected to the filter assembly. Heat the
ingredientes) . dissolution medium to an appropriate temperature taking the
Place the cylinder in the apparatus, and immediately rotate melting point of the preparation into consideration. Using a
the cylinder at 100 r/min, for example. At determined suitable pump, introduce the warmed dissolution medium
intervals, withdraw a sample of dissolution medium from a through the bottom of the cel1 to obtain a suitable
zone midway between the surface of the dissolution medium continuous flow through an open or closed circuit at the
and the top of the rotating cylinder, and not less than 1 cm prescribed rate (± 5 per cent) . When the dissolution
from the vessel wal1. medium reaches the overflow, air starts to escape through the
capillary and chamber B fil1s with the dissolution medium.
Perform the assay on ea eh sample as directed in the
individual monograph, correcting for any volume withdrawn, The preparation spreads through the dissolution medium
according to its physico-chemical properties.
as necessary. Repeat the test with additional patches.
Interpretation The requirements are met if the quantity In justified and authorised cases, representative fractions of
of active ingredientes) released from the patch, expressed as large volume suppositories may be tested.
the amount per surface area per time unit, is within the
prescribed limits at the defined sampling times.
3. Dissolution Test for Lipophilic Solid Dosage

Forros (3)
Apparatus Metal grid
The apparatus (see Figure 2.9.42.-1) consists of:
(2)
- A reservoir for the dissolution medium.
- A pump that forces the dissolution medium upwards
through the flow-through cel1.
- A flow-through cel1 shown in Figure 2.9.42.-2 specifical1y
intended for lipophilic solid dosage forms such as
L{)
suppositories and soft capsules. It consists of 3 transparent L{)
parts which fit into each other. The lower part (1) is made
(1 )
up of 2 adjacent chambers connected to an overflow ce
M
device.
The dissolution medium passes through chamber A and
is subjected to an upwards flow. The flow in chamber B
is downwards directed to a small-size bore exit which
leads upwards to a filter assembly. The middle part (2)
of the cel1 has a cavity designed to collect lipophilic Sieve with
point
excipients which float on the dissolution medium.
A metal grill serves as a rough filter. The upper part (3)
holds a filter unit for paper, glass fibre or cellulose filters. " 1.6
- A water-bath that will maintain the dissolution medium at
37 ± 0.5 oC.
Figure 2.9.42.-2. - Flow-through cel!
Sampling and evaluation

Samples are always collected at the outlet of the cell,
irrespective of whether the circuit is opened or closed.
Filter the liquid removed using an inert filter of appropriate
Reservoir for Pump Thermostatically controJled CoUecting receptades pore size that do es not cause significant adsorption of the
dissolution medium flow-through cell and filter fer analysis
active substance from the solution and do es not contain
substances extractable by the dissolution medium that would
Figure 2.9.42.-1. - Flow-through apparatus
interfere with the prescribed analytical method. Proceed with
analysis of the filtrate as prescribed.
Disso1ution medium If the dissolution medium is The quantity of the active substance dissolved in a specified
buffered, adjust its pH to within ± 0.05 units of the time is expressed as a percentage of the content stated on the
prescribed value. Remove any dissolved gases from the labe!.
dissolution medium before the test since they can cause the
formation of bubbles that significantly affect the results.
Method
Place 1 unit of the preparation to be examined in chamber
A. Close the cel1 with the prepared filter assembly. At the
016 M6x16
o
<O
E
N
070
SECTION G-G
SECTION F-F
• 040
~ F
112
~ __________I
p;-~
~ ~~_~ __~1_21_.4______~
24
A 8 C
A. horizontal piston B. guide C. chewjng chamber D. funnel E. vertical piston
Figure 2.9.25.-1 - Apparatus A - Chewing chamber and pistons

4. Drug Release from Medicated Chewing Gum - 1 central chamber;

(Ph. Eur. method 2.9.25) - 1 fu=el (Figure 2.9.25.-2);
- 2 guides with bushes (Figure 2.9.25 .-3) .
PRINCIPLE
The funnel and guides are mounted on the central chamber.
The test is used to detennine the dissolution rate of active The O-rings are incorporated in the pistan reces s with the
substances in medicated chewing gums. This is done by sealing ring around it; the sealing rings ensure that the
applying a mechanical kneading procedure to a piece of gum chamber is watertight. The horizontal pistons are placed in
placed in a small chamber designed to simulate the process the chewing chamber through the guides.
of chewing.
The gum is artificially chewed by the horizontal pistans, and
APPARATUSA the vertical pistan ensures that the gum stays in the right
place between chews.
Chewing apparatus A (Figure 2.9.25.-1) consists of:
Machine speed is controlled to ensure a constant cyc1e.
- 1 chewing chamber; One cyc1e (chew) is defined as follows: the horizontal pistans
- 1 vertical piston; start from their outennost position, move to their i=ennost
- 2 horizontal pistons with O-rings and sealing rings. position then return ta their outermost position. Within one
The chewing chamber consists of 4 individual parts:
cycle, the vertical piston moves from its lowest position to its APPARATUS B
uppermost position and back to its lowest position. Chewing apparatus B (Figure 2.9 .25.-4) consists of:
Each horizontal piston has a stroke of 25.0 mm. - 1 test cell (Figure 2.9.25 .-5 or 2.9.25.-6);
The maximum distance between these 2 pistons is 50 mm.
- 1 vertical axle with upper chewing surface (Figures
The minimum distance between the 2 horizontal pistons is
2.9.25.-7 and 2.9.25 .-8);
0.1 mm to 1.0 mm. The vertical piston has a stroke of
22.0 mm. - 1 base chamber with lower chewing surface (Figures
2.9.25.-9 and 2.9.25.-10);
Horizontal piston movement is controlled so that the 2
pistons are at their innermost position at the same time. - 1 device for up-and-down chewing motion;
Vertical piston movement is controlled so that it do es not - 1 revolving device for the vertical axle.
conflict with the movement of the horizontal pistons. The gum is artificially chewed by the lower and upper
If necessary, the machine can be constructed so that the chewing surfaces. Machine speed is controlled to ensure a
horizontal pistons rotate around their own axes in opposite constant cycle. The distance between the lower and upper
direction to each other by the end of the chew in order to chewing surfaces may be set up to 5 mm. The turning angle
0
obtain maxirnum chewing. of the revolving device is about 20 •
All parts of the apparatus that may come into contact with
the preparation or the dissolution medium are chemically
inert and do not adsorb, react with or interfere with the
sample.
A
e
Ir
~
"\
..... / JI LT--T..J 1I )
" )::: :::: o
'1" ",'
,," 1 -"V
,, 1 ,...---
11 1 , 1 11
'1' 1 1 1,1
111 1 1 11 1 , ~
'1 1 ,111 /v
'
III.~IIII/
:::.-
(IV
I~V
1, _
~, ...
/
F
Figure 2.9.25.-2 - Funnel tl.J L-"i:1- G
036
A. revolving device for the upper E. upper chewing surface

chewing surface
B. stand F. lower chewing surface
C. test cell G. base chamber
D. axle H. device for up-and-down chewing
motion
Figure 2.9.25.-4 - Apparatus B
The test cells may also be equipped with 1 or 2 glass

Figure 2.9.25.-3 - Cuide (section C-C) sampling tubes, coming through the thermostatic double
Dimensions in millimetres wall. These tubes also make it possible to have an external
059 ± 2
,
/,1
/ I
I
I
I
,
I
I
N
al
Figure 2.9.25.-5 - Test cel!

059±2
N
al
,
I
I
I
I
,
I
I I
I I
'L-.J
t
l. 037.2 ± 0.1
,I
Figure 2.9.25.-6 - Test cel! (straight)

M8
sink, which may be necessary ro achieve sink conditions for
sparingly soluble substances.
The gum is usually sandwiched between 2 circular plastic
nets ro prevent disintegration.
Nets made from nylon (PA6) with an aperture of 1.4 mm
and a wire diameter of 0.405 mm may be used.
All parts of the apparatus that may come into contact with
the preparation or the dissolution medium are chemically
inert and do not adsorb, react with or interfere with the
sample.
M16x1 I
I 11128
1
o 1
N
- I
1
I
1
o
Ol
I
1
I
1
Figure 2.9.25.-8 - Upper chewing surface
¡
~
N
1
fT¡
1 / ~+-Á
'"
61 .6
"'1
¡ ¡
~1
I
M8
08.5
027
Figure 2.9.25.·7 - Axle

PROCEDURE
For each determination, the following information is needed:
- apparatus used (type A or type B);
- composition, volume and temperature of the dissolution
medium;
- number of chews per minute;
- time and sampling method;
- whether the analysis is performed on the gum residue or 037 -SS
on the dissolution medium; 8.8 (2xl N
- method of ahalysis. "1

1 ,08.5 N '"
Place the prescribed volume of dissolution medium in the ___,.
I 11
~
!
11 ...___
chewing chamber, usually 20 mL of phosphate buffer solution
pH 6.0 R2. Maintain the medium temperature at
ro
~ ---t-
===¡
j-~---, 11 , 11 r---....C"1
I 111111 I
1111 II
I !! ' !! I
I
F===
-+----j
o
~
37 ± 0.5 oC using an electrical device with external control
(apparatus A) or with a thermostat (apparatus B). Set the 10
machine speed at the prescribed number of chews per minute

....
(typically up ro 60). Accurately weigh a portion of gum or
the whole gum, put it into the chewing chamber and start the Figure 2.9.25.-9 - Base chamber
machine. Dimensions in millimetres
M8
infiuenced by extrinsic factors (test conditions), such as
hydrodynamics, temperature, viscosity, pH, buffer strength
and ionic strength of the dissolution medium.
The assessment of intrinsic dissolution rate of a solid
substance involves the preparation of a compactoAssurance
of appropriate compaction properties of the powder to be
tested is needed prior to performing the test.
The intrinsic dissolution rate is determined by exposing a
constant area of the compacted substance to an appropriate
dissolution medium, while maintaining constant stirring rate,
temperature, ionic strength and pH.
The intrinsic dissolution rate is expressed in terms of
0 29 dissolved mas s of substance per time per exposed area,
037
typically in milligrams per minute per square centimetre
(mg·min- 1 cm- 2 ).
Blasted surlace RA 1 5-2.1
Apparatus
A typical apparatus consists of a punch and die fabricated
out of hardened steel. The base of the die has 3 threaded
holes for the attachment of a surface pi ate made of polished
steel, providing a mirror-smooth base for the compacto
The die has a 0.1-1.0 cm diameter cavity into which a
measured amount of the powder to be tested is placed.
The punch is then inserted in the die cavity and the material
is compres sed, generally using a benchtop hydraulic press.
A hole through the head of the punch allows insertion of a
metal rod to facilita te removal from the die after the test.
Figure 2.9.25.-10 - Lower chewing surface A compact is formed in the cavity with a single face of
defined area exposed on the bottom of the die (Figure
2.9.29 .-1) . The bottom ofthe die cavity is threaded so that
at least 50-75 per cent of the compact can dissolve without
SAMPLING AND EVALUATION falling out of the die. The top of the die has a threaded
Stop the apparatus at the prescribed time. Remove the gum shoulder that allows it to be attached to a holder. The holder
residue and take a sample of the dissolution medium. is mounw«(on a laboratory stirring device, and the entire die,
Determine the content of active substance(s) by a suitable with the compact still in place, is irnmersed in the dissolution
method. Medium replacement may be made after each medium and rotated by the stirring device .
sampling procedure; compensation by calculation of medium Procedure
volume change or sample dilution is needed. Altematively, Weigh the material onto a piece of weighing paper. Attach
determine the content of active substance(s) remaining in the the surface plate to the underside of the die, and secure it
gum residue. Carry out the test successively on 6 medicated with the 3 provided screws. Transfer the sample of powder
chewing gums. tested into the die cavity. Place the punch into the chamber,
The quantity of active substance(s) dissolved in a specified and secure the metal plate on the top of the assembly.
time is expressed as a percentage of the content stated on the Compress the powder using a hydraulic press by applying a
label. suitable pressure for a sufficient dwell time to ensure a stable
compact with minimal porosity; the disintegration of the
5. Intrinsic Dissolution compact has to be prevented as far as possible, since it would
(Ph. Eur. method 2.9.29) cause an increase in surface area and hence in dissolution
The test is intended to determine the intrinsic dissolution rate. Detach the surface plate, and screw the die with punch
rate of pure solid substances following compaction. It is still in place into the holder. Tighten securely. Remove all
carried out under specified experimental conditions such that loose powder from the surface of the die by blowing
a practicar measure of the intrinsic dissolution rate is compressed air or nitrogen across the surface of the compact.
obtained. Slide the die-holder assembly into the dissolution test chuck
The intrinsic dissolution rate is a theoretical value referring to and tighten. Position the shaft in the spindle so that when
pure solid substances having null porosity, but, practically, the test head is lowered, the exposed surface of the compact
intrinsic dissolution rate is determined on substances having will be 3.8 cm from the bottom ofthe vessel. The disc
a minhnal porosity. assembly is aligned to minimise wobble and air bubbles are
not allowed to form as this could decrease the compact
PrincipIe surface in contact with the dissolution medium. If possible,
The intrinsic dissolution rate is defined as the dissolution rate sink conditions are maintained throughout the test. However,
of pure substances following compaction under the condition in order to obtain detectable concentrations of solute, the use
of constant surface area. Its assessment is useful in the of a relatively small volume of medium may be necessary as a
characterisation of active substances and excipients. consequence of the limited surface available for dissolution.
The dissolution rate of pure substances can be affected by all Warm the dissolution medium to the temperature chosen for
the solid state properties such as crystal habit, crystallinity, the test. Lower the test head into position before rotation.
amorphism, polymorphism, pseudo-polymorphism, particle Care should be taken to ensure that air bubbles are excluded
size and specific surface area. In addition, it can also be
09 .75 ± 0.35 F
041.1 ± 0.4
o 8.0 - t - - - . (
E
A
101
I~ ~I
- ,--.---------------....,
054 .0 ± 0.4 ¡..----I-++---I B
--~-------------~
07.985 ± 0.005
L 1.-----_ _------, A
;¡
A. Surface plate C. Neoprene gasket E. Holder and shaft assembly
B. Die D. Punch F. Die underside
,
Figure 2.9.29.·1. - Typical apparatus used to obtain the compact for the determination of the infrinsic dissolution
from the surface of the compact as this could de crease the perforrned on the norrnalised experimental data relevant to
compact surface in contact with the dissolution medium. an apprapriate time interval preceding the possible
Operate the apparatus immediately at the speed of rotation disintegration of the compacto The intrinsic dissolution rate
chosen for the test. of the substance tested, expressed in milligrams per minute
Collect samples at fixed time intervals and assay them by per square centimetre, is deterrnined fram the slope of the
means of an analytical methód of suitable sensitivity and regression line. The result for intrinsic dissolution rate must
accuracy. be accompanied by a statement of the precise conditions of
compact preparation and test method (dissolution medium,
Assessment 01 the results volume of medium ~sed, stirring rate, temperature etc.).
The data for the cumulative amount dissolved at each time NOTE: when necessary and justified, an apparatus with a
point are corrected for sampling losses. To calculate the
different configuration may be used, such as a die holder that
intrinsic dissolution rate, plot the cumulative amount of holds the compact in a fixed vertical position, with agitation
sample dissolved per unit area of the compact against time. provided by a paddle positioned at a defined distance from
The cumulative amount dissolved per unit area is given by
the surface of the compacto
the cumulative amount dissolved at each time point divided
by the surface area exposed. Linear regression is then
A B e o
A. reservo ir lor dissolution medium B. pump C. thermostatically controlled f1ow·through cell and filter D. collecting vessels for analysis
Figure 2.9.43.·1. - Flow-through apparatus
6. Apparent Dissolution dissolution medium before the test, since they can cause the
(Ph. Eur. methad 2.9.43) formation of bubbles, which significantly affect the results.
This method is mainly used to determine the apparent Method
dissolution rate of pure solid substances. It may also be used Place a bead of 5 ± 0.5 mm diameter at the bottom of the
for the determination of the apparent dissolution rate of cone of the lower part followed by glass beads of suitable
active substances in preparations presented as powders or size, preferably of 1 ± 0.1 mm diameter. Place a sieve (with
granules. 0.2 mm ,apertures), a suitable filter and a 2nd sieve on top of
Apparatus the lower part. Fit the middle part onto the lower part.
AlI parts of the apparatus that may come into contact with Weigh the assembly. Place the sample on the filtration
the sample or the dissolution medium are chemically inert assembly and weigh the sample in the cell. Place the sieve of
and do not adsorb, react with, or interfere with the test the insert, cone upwards, on the sample, and position the
sample. No part of the assembly or its environment clip midway down the middle parto Place a sieve (with
contributes significant motion, agitation or vibration beyond 0.2 mm apertures) and a suitable filter on top of the middle
that resulting from the fiow-through system. part. Fit the upper part. Heat the dissolution medium to the
chosen temperature . Using a suitable pump, introduce the
Apparams that permits observation of the sample is
dissolution medium through the bottom of the cell to obtain
preferable:
a suitable continuous fiow through an open or closed circuit
The apparatus (see Figure 2.9.43.-1) consists of: at the prescribed rate ± 5 per cent.
- a reservoir for the dissolution medium;
Sampling
- a pump that forces the dissolution medium upwards
Samples óf dissolution medium are collected at the outlet of
through the fiow-through celli
the cell, irrespective of whether the circuit is opened or
- a fiow-through cell, preferably of transparent material, closed.
mounted vertically with a filter system preventing escape
Immediately filter the liquid removed using an inert filter of
of undissolved particlesi
appropriate pore size that does not cause significant
- a water-bath that will maintain the dissolution medium at adsorption of the substances from the solution and does not
the chosen temperamre (generally 37 ± 0.5 OC). contain substances extractable by the dissolution medium
The fiow-through cell shown in Figure 2.9.43.-2 consists of that would interfere with the prescribed analytical method.
3 parts that fit into each other. The lower part supports a Proceed with the analysis of the filtrate as prescribed.
system of grids and filters on which the powder is placed.
The middle part, which fits onto the lower part, contains an Assessment of the results
insert that sieves the sample when the dissolution medium When the test is performed for batch releas e purposes, an
fipws through the cell. This insert is made up of 2 parts: a adequate number of replícates is carried out.
conical sieve that is placed on the sample and a clip placed The results are expressed as:
midway down the middle part to hold the sieve in place
when the dissolution medium passes through. A 2nd filtration - the amount of dissolved substance by time unit (if the
assembly (grid and filter) is placed on top of the middle part dissolution is linear);
before fitting the upper part through which the dissolution - the dissolution time of the whole sample and at
medium fiows out of the cel!. appropriate intermediate stages.
Dissolution medium
If the dissolution medium is buffered, adjust its pH to within
± 0.05 units. Remove any dissolved gases from the
2014 Appendix XII e V-A357
Table 2.9.5.-1
Pharmaceutical Form Average Mass Percentage
~ __ F deviation
Tablets (uncoated and 80 mg or less 10
film-coated)
More than 80 mg and
7.5
less than 250 mg
250 mg or more 5
~_ _ _ _ E Capsules, granules (uncoated, Less than 300 mg 10
single-dose) and powders
(single-dose) 300 mg or more 7.5
c:o /'"""___ c
LO Powders for parenteral More than 40 mg 10
administration* (single-dose)
Suppositories and pessaries Al! masses 5
Powders for eye-drops and Less than 300 mg 10

C"l /,"""_ _ _ B powders for eye lotions 300 mg or more 7.5
C"l (single-dose)
N
* When the average mass is equal to or below 40 mg, the preparation
T'" is not submitted to the test for uniformity of mass but to the test for
T'"
uniformity of content of single-dose preparations (2.9.6).
Powders ¡or Parenteral Administration

Remove any paper labels from a container and wash and dry
the outside. Open the container and without de/ay weigh the
0.8 container and its contents. Empty the container as
completely as possible by gentle tapping, rinse it if necessary
with water R and then with alcohol R and dry at 100-105 °e
for 1 h, or, if the nature of the container precludes heating at
A. lower part C. clip E. middle part this temperature, dry at a lower temperature to constant
mass. Allow to cool in a desiccator and weigh. The mass of
B. sieve D. insert F. upper part the contents is the difference between the weighings. Repeat
the procedure with another 19 containers.
Figure 2.9.43.-2. - Flow-through cel!
2. Uniformity ofWeight (Mas s) ofDelivered Doses
Dimensions in millimetres from Multidose Containers
(Ph . Eur. method 2.9.27)
The following test is intended for oral dosage forms such as
granules, powders for oral use and liquids for oral use, which are
supplied in multidose containers provided at manufacture with a
C. Consistency of Formulated measuring device.
Preparations Weigh individually 20 doses taken at random from one or
more containers with the measuring device provided and
1. Uniformity ofWeight (Mass) determine the individual and average masses. Not more than
(Ph. Eur. method 2.9.5) 2" of the individual mas ses deviate from the average mass by
Weigh individually 20 units taken at random or, for single- more than 10 per cent and none deviates by more than
dose preparations presented in individual containers, the 20 per cent.
contents of 20 units, and determine the average mass.
Not more than 2 of the individual masses deviate from the
3. Uniformity of Content
average mas s by more than the percentage deviation shown (Ph. Eur. method 2.9.6)
in Table 2.9.5.-1 and none deviates by more than twice that The test for uniformity of content of single-dose preparations
percentage. is based on the assay of the individual contents of active
For capsules and powders for parenteral administration, substance(s) of a number of single-dose units to determine
proceed as described be/ow. whether the individual contents are within limits set with
reference to the average content of the sample.
Capsules
The test is not required for multivitamin and trace-element
Weigh an intact capsule. Open the capsule without losing any preparations and in other justified and authorised
part of the shell and remQve the contents as completely as drcumstances.
possible. For soft shell capsules, wash the shell with a
Method Using a suitable analytical method, determine the
suitable solvent and allow to stand until the odour of the
individual contents of active substance(s) of 10 dosage units
solvent is no longer perceptible. Weigh the shell. The mas s of
taken at random.
the contents is the difference between the weighings. Repeat
the procedure with another 19 capsules. Apply the criteria of test A, test B or test e as specified in
the monograph for the dosage form in question.
TestA
Tablets, powders ¡or parenteral administration,
ophthalmic inserts, suspensions ¡or injection The
preparation complies with the test if each individual content
is between 85 per cent and 115 per cent of the average
V-A358 Appendix XII e 2014
Table 2.9.40.-1. - Applicatian af Cantent Unifarmity (CU) and Mass Variatian (MV) test far dasage farms
Dosage forms Type Sub-Type Dose and ratio of active substance
~ 25 mg and ;, 25 per cent < 25 mg or < 25 per cent
Tablets uncoated MV CU
coated film-coated MV CU
others CU CU
Capsules hard MV CU
soft suspensions, emulsions, gels CU CU
solutions MV MV
Solids in single-dose MV MV
single component
containers
solution freeze-dried in final MV MV

multiple components container
others CU CU
Solutions encJosed in MV MV
single-dose containers
Others CU CU
contento The preparation fails to comply with the test if between 75 per cent and 125 per cent of the average
more than one individual content is outside these limits or if contento
one individual content is outside the limits of 75 per cent to
125 per cent of the average contento 4. UniforIlÚty of Dosage Units
If one individual content is outside the limits of 85 per cent (Ph. Eur. Method 2.9.40)
to 115 per cent but within the limits of 75 per cent to To ensure the consistency of dosage uruts, each unit in a
125 per cent, determine the individual contents of another batch should have an active substance content within a
20 dosage uruts taken at random. The preparation complies narrow range around the label claim. Dosage units are
with the test if not more than one of the individual contents defined as dosage forms containing a single dose or a part of
of the 30 units is outside 85 per cent to 115 per cent of the adose of an active substance in each dosage unit. Unless
average content and none is outside the limits of 75 per cent otherwise stated, the uniformity of dosage units specification
to 125 per cent of the average contento is not intended to apply to suspensions, emulsions or gels in
TestB single-dose containers intended for cutaneous administration.
The test for content uniformity is not required for
Capsules, powders other than lor parenteral
multivitamin and trace-element preparations.
administration, granules, suppositories, pessaries
The preparation complies with the test if not more than one The term 'uniformity of dosage unit' is defined as the degree
individual content is outside the limits of 85 per cent to of uniformity in the amount of the active substance among
115 per cent of the average content and none is outside the dosage uruts. Therefore, the requirements of this chapter
limits of 75 per cent to 125 per cent of the average contento apply to each active substance being comprised in dosage
The preparation fails to comply with the test if more than 3 units containing one or more active substances, unless
individual contents are outside the limits of 85 per cent to otherwise specified elsewere in this Pharmacopoeia.
115 per cent of the average content or if one or more The uniformity of dosage units can be demonstrated by
individual contents are outside the limits of 75 per cent to either of 2 methods: content uniformity or mass variation
125 per cent of the average contento (se e Table 2.9.40.-1 ).
If 2 or 3 individual contents are outside the limits of The test for content uniformity of preparations presented in
85 per cent to 115 per cent but within the limits of dosage units is based on the assay of the individual contents
75 per cept to 125 per cent, determine the individual of active substance(s) of a number of dosage units to
contents of another 20 dosage uruts taken at random. determine whether the individual contents are within the
The preparation complies with the test if not more than 3 limits set. The content uniformity method may be applied in
individual contents of the 30 units are outside the limits of all cases.
85 per cent to 115 per cent of the average content and none The test for mass variation is applicable for the following
is outside the limits of 75 per cent to 125 per cent of the dosage forms:
average contento (1 ) solutions enclosed in single-dose containers and in soft
Test C capsules;
Transdermal patches The preparation complies with the (2) solids (including powders, granules and sterile solids)
test if the average content of the 10 dosage units is between that are packaged in single-dose containers and contain
90 per cent and 110 per cent of the content stated on the no added active or inactive substances;
label and if the individual content of each dosage unit is
Table 2.9.40.-2.
Variable Definitíon Condítíons Value
X Mean oC individual contents (xl'

x2• ... x,). expressed as a percentage
of the label cIaim
x 1, XZ, ,,,,xl! Individual contents of the

dosage units tested. expressed
as a percentage of the label cIaim
n Sample size (number of dosage units

in a sample)
k Acceptability constant If n = 10, then 2.4
If n = 30, then 2.0
s Sample standard deviation
[" T'
i~l (Xi -
n - 1
X)
RSD Relative standard deviation IDOs

X
M (case 1) Reference value lf 98.5 per cenl ,; X ,; 101.5 per M=X

To be applied when r,; 101.5 cent. then (AV= ks)
If X < 98.5 per cent, then M = 98.5 per cent

(AV= 98.5 - X + ks)
lf X> 101.5 per cent, then M = 101.5 per cent

(AV= X - 101.5 + ks)
M (case 2) Reference value lf 98.5 per cent ,; X ,; r, then M= X

To be applied when r> 101.5 (AV= ks)
If X < 98.5 per cen t, then M = 98.5 per cent

(AV= 98.5 -X + ks)
If X> r, then M = r per cent

(AV=X-T+ks)
Acceptance value (A V) General formu la:

1M - XI + ks
CaJculations are specified aboye
for the different cases.
LI Maximum aIlowed acceptance value Ll = 15.0 unless otherwise specified
L2 Maximum aIlowed range for deviation On the low side, no dosage unit result L2 = 25.0 unless otherwise specified
of each dosage unit tested from can be less than 0.75 M while on
the calculated. value of M the high side, no dosage unit result
can be greater than 1.25 M (This is
based on L2 value of 25.0)
r Target content per dosage unit at

. time of manufacture, expressed as
a percentage of the label cIaim.
Unless otherwise stated, T is
equal to 100 per cent or T is the
manufacturer's approved target
content per dosage unit
(3) solids (including sterile solids) that are packaged in substances present in lesser proportions is demonstrated
single-dose containers, with or without added active or by meeting content uniformity requirements.
inactive substances, that have been prepared from true The test for content uniformity is required for al! dosage
solutions and freeze-dried in the final containers and are forms not meeting the aboye conditions for the mass
label!ed to indica te this method of preparation; variation test. Altematively, products that do not meet the
(4) hard capsules, uncoated tablets, or film-coated tablets, 25 mg!25 per cent threshold limit may be tested for
containing 25 mg or more of an active substance uniformity of dosage units by mass variation instead of the
comprising 25 per cent or more, by mass, of the dosage content uniformity test on the fol!owing condition: the
unit or, in the case of hard capsules, the capsule concentration Relative Standard Deviation (RSD) of the
contents, except that uniformity of other active active substance in the final dosage units is not more than
2 per cent, based on process validation data and development of product removed from rhe individual capsules and the
data, and if there has been regulatory approval of such a result of the assay. Calculate the acceptance value.
change. The concentration RSD is rhe RSD of rhe Solid dosage fonns other than tablets and capsules
concentration per dosage unit (m/m or m/ V), where Proceed as directed for hard capsules, treating each unit as
concentration per dosage unit equals rhe assay result per described therein. Calculate the acceptance value.
dosage unit divided by rhe individual dosage unit mass.
Liquid or semi-solid dosage fonns Accurately weigh the
See rhe RSD formula in Table 2.9.40.-2. amount of liquid or semi-solid that is removed from each of
10 individual containers in conditions of normal use .
CONTENT UNIFORMITY
If necessary, compute rhe equivalent volume after
Select not fewer rhan 30 units, and proceed as follows for rhe determining the density. Calculate the active substance
dosage form designated. Where different procedures are used content in each container from the mass of product removed
for assay of rhe preparation and for rhe content uniformity from the individual containers and the result of the assay.
test, it may be necessary to establish a correction factor to be Calculate the acceptance value.
applied to rhe results of rhe latter.
Calculation of Acceptance Value Calculate the
Solid dosage fonns Assay 10 units individually using an acceptance value (A V) as shown in content uniformity,
appropriate analytical method. Calculate rhe acceptance except that the individual contents of the units are replaced
value (see Table 2.9.40.-2). with the individual estimated contents defined below.
Liquid or semi-solid dosage fonns Assay 10 units individual estimated contents of the dosage
individually using an appropriate analytical method. Carry units tested;
out the assay on the amount of well-mixed material that is
removed from an individual container in conditions of where
normal use . Express rhe results as delivered dose. Calculate
A
the acceptance value (see Table 2.9.40.-2). Xi =Wi X
W
Calculation of Acceptance Value
Calculate the Acceptance Value (A V) using the formula: W¡, Wz, ... , W n individual masses of the dosage units
tested;
1M-xl +ks A content of active substance (percentage of
label claim) obtained using an appropriate
analytical method (assay);
for which the terms are as defined in Table 2.9 .40 .-2.
W mean of individual masses (w ¡, Wz, ... , wJ.
MASS VARIAnON CRITERIA
Carry out an assay for the active substance(s) on a
Apply the following criteria, unless otherwise specified.
representative sample of the batch using an appropriate
analytical method. This value is result A, expressed as Solid" semi-solid and liquid dosage fonns The
percentage of label c1aim (se e Calculation of Acceptance requirements for dosage uniformity are met if the acceptance
Value). Assume that the concentration (mass of active value of the first 10 dosage units is less than or equal to
substance per mas s of dosage unit) is uniformo Select not L1 per cent. If the acceptance value is greater than
fewer than 30 dosage units, and proceed as follows for the L1 per cent, test the next 20 dosage units and calculate the
dosage form designated. acceptance value. The requirements are met if the final
acceptance value of the 30 dosage units is les s than or equal
Uncoated or film-coated tablets Accurately weigh 10
to L1 per cent and no individual content of the dosage unit
tablets individually. Calculate the active substance content,
is les s than (1 -L2 x O.Ol)M or more than (1 + L2 x
expressed as percentage of label c1aim, of each tablet from
O.Ol)M in calculation of acceptance value under content
the mass of the individual tablets and the result of the assay.
uniformity or under mass variation. Unless otherwise
Calculate the acceptance value.
specified, L1 is 15.0 and L2 is 25.0.
Hard capsules Accurately weigh 10 capsules individually,
taking care to preserVe the identity of each capsule. Remove 5. Extractable Volume ofParenteral Preparations¡
the contents of each capsule by suitable means. Accurately (Ph. Eur. method 2.9.17)
weigh the emptied shells individually, and calculate for each Suspensions and emulsions are shaken before withdrawal of
capsule the net mass of its contents by subtracting the mass the contents and before the determination of the density.
of the shell from the respective gross mass. Calculate the Oily and viscous preparations may be warmed according to
active substance content in each capsule from the mas s of the instructions on the label, if necessary, and thoroughly
product removed from the individual capsules and the result shaken immediately before removing rhe contents.
of the assay. Calculate the acceptance value. The contents are then cooled to 20-25 oC before measuring
Soft capsules Accurately weigh 10 intact capsules the volume.
individually to obtain their gross masses, taking care to
Single-dose containers
preserve the identity of each ,capsule. Then cut open the
capsules by means of a suitable c1ean, dry cutting instrument Select 1 container if the nominal volume is 10 mL or more,
such as scissors or a sharp open blade, and remove the 3 containers if the nominal volume is more than 3 mL and
contents by washing with a suitable solvent. Allow the less than 10 mL, or 5 containers if the nominal volume is
occ1uded solvent to evaporate from rhe shells at room 3 mL or less. Take up individually the total contents of each
temperature over a period of about 30 min, taking container selected into a dry syringe of a capacity not
precautions to avoid uptake or los s of moisture. Weigh the exceeding 3 times the volume to be measured, and fined
individual shells, and calculate the net contents. Calculate
1 This Chapter has undergone pharmacopoeial harmonisation. See chapter
the active substance content in each capsule from the mass
5.8. Pharmacopoe'ial hannonisation.
with a 21-gauge needle not les s than 2.5 cm in length. Expel from a volatile solvent. Plate coating must be part of method
any air bubbles from the syringe and needle, then discharge validation and may be omitted where justified and
the contents of the syringe without emptying the needle inta authorised.
a standardised dry cylinder (graduated ta contain rather than Mass balance The total mas s of the active substance is
to deliver the designated volumes) of such size that the not less than 75 per cent and not more than 125 per cent of
volume ta be measured occupies at least 40 per cent of its the average delivered dose determined during testing for
graduated volume. Alternatively, the volume of the contents uniformity of delivered dose. This is not a test of the inhaler
in millilitres may be calculated as the mass in grams divided but it serves to ensure that the results are valid.
by the density.
For containers with a nominal volume of 2 mL or les s, the Apparatus A - Glass impinger
contents of a sufficient number of containers may be pooled The apparatus is shown in Figure 2.9.18.-1 (see also
ta obtain the volume required for the measurement provided Table 2.9.18.-1).
that a separate, dry syringe assembly is used for each
Pro ce dure lar nebulisers
container. The contents of containers holding 10 mL or
more may be determined by opening them and emptying the Introduce 7 mL and 30 mL of a suitable solvent into the
contents directly into the graduated cylinder or tared beaker. upper and lower impingement chambers, respectively.
The volume is not less than the nominal volume in case of Connect all the component parts. Ensure that the assembly is
containers examined individually, or, in case of containers vertical and adequately supported and that the jet spacer peg
with a nominal volume of 2 mL or less, is not less than the of the lower jet assembly just touches the bottom of the
sum of the nominal volumes of the containers taken lower impingement chamber. Connect a suitable pump fitted
collectively. with a filter (of suitable pore size) ta the outlet of the
apparatus. Adjust the air flow through the apparatus, as
Multidase cantainers measured at the inlet 10 the throat, to 60 ± 5 Umin.
For injections in multidose containers labelled ta yield a Introduce the liquid preparation for inhalation into the
specific number of doses of a stated volume, select one reservoir of the nebuliser. Fit the mouthpiece and connect it
container and proceed as directed for single-dose containers by means of an adapter to the device.
using the same number of separate syringe assemblies as the
Switch on the pump of the apparatus and after lOs switch
number of doses specified.
on the nebuliser.
The volume is such that each syringe delivers not less than
After 60 s, unless otherwise justified, switch off the nebuliser,
the stated dose.
wait for about 5 s and then switch off the pump of the
Cartridges and prefilled syringes apparatus. Dismantle the appararus and wash the inner
Select 1 container if the nominal volume is 10 mL or more, surface of the upper impingement chamber collecting the
3 containers if the nominal volume is more than 3 mL and washings in a volumetric flask. Wash the inner surface of the
less than 10 mL, or 5 containers if the nominal volume is lower impingement chamber collecting the washings in a
3 mL or less. If necessary, fit the containers with the second volumetric flask. Finally, wash the filter preceding the
accessories required for their use (needle, piston, syringe) and pump and its connections 10 the lower impingement chamber
transfer the entire contents of each container without and combine the washings with those obtained from the
emptying the needle into a dry tared beaker by slowly and lower impingement chamber. Determine the amount of
constantly depressing the pistan. Determine the vol~me in active substance collected in each of the 2 flasks. Express the
millilitres calculated as the mas s in grams divided by the results for each of the 2 parts of the apparatus as a
density. percentage of the total amount of active substance.
The volume measured for each of the containers is not less Pro ce dure lar pressurised inhalers
than the nominal volume. Place the actua10r adapter in position at the end of the throat
Parenteral infusians so that the mouthpiece end of the actua1Or, when inserted to
Select one container. Transfer the contents inta a dry a depth of about 10 mm, lines up along the horizontal axis of
measuring cylinder of such a capacity that the volume to be the throat and the open end of the actua1Or, which accepts
determined occupies at least 40 per cent of the nominal the pressurised container, is uppermost and in the same
volume of the cylinder. Measure the volume transferred. vertical plane as the rest of the apparatus.
The volume is not less than the nominal volume. In't roduce 7 mL and 30 mL of a suitable solvent into the
upper and lower impingement chambers, respectively.
7. Preparations for Inhalation: Aerodynamic Connect all the component parts. Ensure that the assembly is
Assessment of Fine Particles vertical and adequately supported and that the lower jet-
(Ph. Eur. methad 2.9.18) spacer peg of the lower jet assembly just touches the bottom
This test is used to determine the fine particie characteristics of the lower impingement chamber. Connect a suitable pump
of the aerosol ciouds generated by preparations for 10 the outlet of the apparatus. Adjust the air flow through the
inhalation. apparatus, as measured at the inlet 10 the throat, to 60 ± 5
Umin.
Unless otherwise justified and authorised, one of the
following apparatus and test procedures is used. Prime the metering valve by shaking for 5 s and discharging
once to waste; after not less than 5 s, shake and discharge
Stage mensuratian is performed periodically together
again 10 waste. Repeat a further 3 times .
with confirmation of other dimensions critical ta the effective
operation of the impactar.
Re-entrainment (far apparatus D and E) To ensure
efficient particie capture, coat each plate with glycerol,
silicone oil or similar high viscosity liquid, typically deposited
Table 2.9.18.-1. - Component specification for apparatus A 95

in Figure 2.9.18.-1
Code Item Description Dimen-
sions· --~~A
A Mouthpiece Moulded rubber adapter for actuator
adaptor mouthpiece.
B Throat Modified round-bottomed flask: 50mL 707
- ground-g/ass in/el sockel 29/32
C Neck
- ground-g/ass oul/el cone
Modified glass adapter:
- ground-g/ass in/el sockel
24/ 29
24/29
45 "
c:::::, -- - - --
!
-'-
40
- ground-g/ass oul/el cone 24/ 29

Lower outlet section of precision-bore
glass tubing:
- bore diameler 14
Selected bore light-wall glass tubing: 75
= - - - -- --'-
- exlerna/ diameler 17
O Upper Modified round·bottomed f1ask 100 mL
~G
impingement - ground-g/ass in/el sockel 24/29
chamber - ground-g/ass out/el cone 24/29
E Coupling tube Medium·wall glass tubing :
.... _05.3
- ground-g/ass cone 14/ 23
G'~
Bent section and upper vertical
section :
- externa/ diameler 13
Lower vertical section:
- exlerna/ diameler 8 07.85 ± 0.725
F Screwthread, Plastic screw cap 28/ 13

Figure 2.9.18.-1. - Apparatus A: glass impinger
side-arm Silicone rubber ring 28/11 Dimensions in millimetres (tolera n ces ± 1 mm unless
adaptor PTFE washer 28/11 otherwise prescribed)
Glass screwthread:
Wash the inner surface of the inlet tube to the lower
- Ihread size 28
impingement chamber and its outer surface that projects into
Side-arm outlet to vacuum pump: the chamber with a suitable solvent, collecting the washings
- minimum bore diameler 5 in the lower impingement chamber. Determine the content
see
of active substance in this solution. Calculate the amount of
G Lower jet Modified polypropylene filter holder
assembly connected to lower vertical section of Figure active substance collected in the lower impingement chamber
coupling tube by PTFE tubing. 2.9.18.-1 per discharge and express the results as a percentage of the
Acetal circular disc with the centres dose stated on the labe!'
of four jets arranged on a projected
circle of diameter 5.3 mm with an Procedure lor powder inhalers
integral jet spacer peg: 10
- peg diameler 2
Introduce 7 mL and 30 mL of a suitable solvent into the
upper and lower impingement chambers, respectively.
- peg prolrusion 2
Connect all the component parts. Ensure that the assembly is
H Lower Conical flask 250mL vertical and adequately supported and that the jet-spacer peg
impingement - ground-g/ass in/el sockel 24/ 29 of the lower jet assembly just touches the bottom of the
chamber lower impingement chamber. Without the inhaler in place,
• Dimensions in millimetres, unless otherwise stated. connect a suitable pump to the outlet of the apparatus .
Adjust the air fiow through the apparatus, as measured at the
inlet to the throat, to 60 ± 5 Umin.
Shake for about 5 s, switch on the pump to the apparatus
Prepare the inhaler for use and locate the mouthpiece in the
and locate the mouthpiece end of the actuator in the adapter,
apparatus by means of a suitable adapter. Switch on the
discharge once immediately. Remove the assembled inhaler
pump for 5 s. Switch off the pump and remove the inhaler.
from the adapter, shake for not les s than 5 s, relocate the
Repeat the discharge sequence. The number of discharges
mouthpiece end of the actuator in the adapter and discharge
should be minimised and typically would not be greater than
again. Repeat the discharge sequence. The number of
10. Dismande the apparatus.
discharges should be minimised and typically would not be
greater than 10. After the final discharge wait for not less Wash the inner surface of the inlet tube to the lower
than 5 s and then switch off the pump. Dismantle the impingement chamber and its outer surface that projects into
apparatus. the chamber with a suitable solvent, collecting the washings
in the lower impingement chamber. Determine the content
of active substance in this solution. Calculate the amount of
2014 Appen dix XII e V-A363
active substance collected in the lower impingement chamber Table 2.9.18.-2. - Component specirication ro r apparatus C
per discharge and express the results as a percentage of the in Figures 2.9.18.-4/ 6
Code' Item Description Dimen-
dos e stated on the labe!' sions··
A,H Jet tube Metal tube screwed onto partition see Figure
Fine particle dos e and p ar ticle size distribution wall sealed by gasket (C), polished 2.9.18.-5
inner surface
Apparatus e - Multi-stage liquid impinger B,G Partition wall Circular metal plate
The multi-stage liquid impinger consists of impaction stages - diameler 120

1 (pre-separator), 2, 3 and 4 and an integral filter stage - lhickness see Figure
(stage 5), see Figures 2.9.18.-4/6. An impaction stage 2.9.18.-5
comprises an upper horizontal metal partition wall (B) e Gasket e.g. PTFE to fit jet
tube
through which a metal inlet jet tube CA) with its impaction O Impaction Porosity Osintered-glass disk
plate (D) is protruding. A glass cylinder (E) with sampling
plate - diameler see Figure
port (F) forms the vertical wall of the stage, and a lower 2.9.18.-5
horizontal metal partition wall (G) through which the tube E Glass cylinder Plane polished cut glass tube
(H) connects to the next lower stage. The tube into stage 4
- heighl, including gaskels 46
(U) ends in a multi-jet arrangement. The impaction plate
(D) is secured in a metal frame (J) which is fas tened by 2 - ouler diameler 100
wires (K) to a sleeve (L) secured on the jet tube. - wall lhickness 3.5
The horizontal face of the collection plate is perpendicular to
- sampling porl (F) diameler 18
the axis of the jet tube and centrally aligned. The upper
surface of the impaction plate is slight1y raised above the - stopper in sampling port ISO 24/ 25
edge of the metal frame. A reces s around the perimeter of J Metal frame lrprofiled circular frame with slit
the horizontal partition wall guides the position of the glass
- inner diameler to fit
cylinder. The glass cylinders are sealed against the horizontal impaction
partition walls with gaskets (M) and c1amped together by 6 plate
bolts eN) . The sampling ports are sealed by stoppers. - heighl 4
The bottom-side of the lower partition wall of stage 4 has a - thickness of horizontal section 0.5
concentrical protrusion fitted with a rubber O-ring (P) which
- thickness of vertical secUon 2
seals against the edge of a filter placed in the filter holder.
K Wire Steel wire interconnecting metal
frame and sleeve (2 for each frame)
- diameter 1
Q
L Sleeve Metal sleeve secured on jet tube by
B screw
- inner diameter to fitjet
tube
- height 6
Stage 1 - thickness 5
(pre-separa tor)
M Gasket e.g. silicone to fit glass
cvlinder
N Bolt Metal bolt with nut (6 pairs)
- length 205
Stage 2 - diameter 4
P O-ring Rubber O-ring
- diameler x thickness 66.34 x 2.62
Q O-ring Rubber O-ring
- diameter x thickness 29.1 x 1.6
Stage 3
R Filter holder Metal housing with stand and oullet see Figure
2.9.18.-6
S Filter support Perforated sheet metal
- diameter 65
Stage 4 - hale diameler 3
- dislance between hales 4

(cenlre-points)
Stage 5 (filter) T Snap-Iocks
U Multi-jet tube Jet tube (H) ending in multi-jet see inserts
arrangement. Figure
2.9.18.-5
Outlet
, Refers to Figure 2.9.18.-4.
•• Measures in millimetres with toleran ces according to iso 2768-m
unless otherwise stated.
Figure 2.9.18.-4. - Apparatus C: multi-stage [iquid impinger

stage center~ ;
t I
s
6x)
v H
k <O
j (7x)
h (7x)
K N
multi-jet pattern
multi-jet U only
D
d
Figure 2.9.18.-5. - Apparatus C: details ofjet tube and impaction plateo Inserts show end of multi-jet tube U leading to stage 4.
(Numbers and lowercase lefters refer to Table 2.9.18.-3 and uppercase letters refer to Figure 2.9.18.-4).
The filter holder (R) is constructed as a basin with a assembled inhaler from the adapter. Repeat the procedure.
concentrical reces s in which a perforated filter support (S) is The number of discharges should be minimised and typically
flush-fitted. The filter holder is dimensioned for 76 mm would not be greater than 10. The number of discharges is
diameter filters. The assembly of impaction stages is clamped suffi.cient to ensure an accurate and precise determination of
onto the filter holder by 2 snap-Iocks (T). Connect an the fine particle dose. After the final discharge, wait for 5 s
induction port (see Figure 2.9.18.-7) onto the stage 1 inlet and then switch off the pump.
jet tube of the impinger. A rubber O-ring on the jet tube Dismantle the filter stage of the apparatus. Carefully remove
provides an airtight connection to the induction porto the filter and extract the active substance into an aliquot of
A suitable mouthpiece adapter is used to provide an airtight the solvento Remove the induction port and mouthpiece
seal between the inhaler and the induction port. The front adapter from the apparatus and extract the active substance
face of the inhaler mouthpiece must be flush with the front into an aliquot of the solvento If necessary, rinse the inside of
face of the induction port. the inlet jet tube to stage 1 with solvent, allowing the solvent
Pro ce dure for pressúrised inhalers to flow into the stage. Extract the active substance from the
Dispense 20 mL of a solvent, capable of dissolving the active inner walls and the collection plate of each of the 4 upper
substance into each of stages 1 to 4 and replace the stoppers. stages of the apparatus into the solution in the respective
Tilt the apparatus to wet the stoppers, thereby neutralising stage by carefully tilting and rotating the apparatus, observing
electrostatic charge. Place a suitable filter capable of that no liquid transfer occurs between the stages.
quantitatively collecting the active substance in stage 5 and Using a suitable method of analysis, determine the quantity
assemble the apparatus. Place a suitable mouthpiece adapter of active substance contained in each of the aliquots of
in position at the end of the induction port so that the solvent.
mouthpiece end of the actuator, when inserted, lines up Calculate the fine particle dose (see Calculations).
along the honzontal axis of the induction port and the
Procedure for powder inhalers
inhaler is positioned in the same orientation as intended for
use. Connect a suitable vacuum pump to the outlet of the Place a suitable low resistance filter capable of quantitatively
apparatus and adjust the air flow through the apparatus, as collecting the active substance in stage 5 and assemble the
measured at the inlet to the induction port, to 30 Umin apparatus. Connect the apparatus to a flow system according
(± 5 per cent). Switch off the pump. to the scheme specified in Figure 2.9.18.-8 and
Table 2.9 .18.-4. Unless otherwise defined, conduct the test
Unless otherwise prescribed in the patient instructions, shake
at the flow rate, Qou" used in the test for uniformity of
the inhaler for 5 s and discharge 1 delivery to waste. Switch delivered dose, drawing 4 L of air from the mouthpiece of
on the pump to the apparatus, locate the mouthpiece end of the inhaler and through the apparatus.
the actuator in the adapter and discharge the inhaler into the
apparatus, depressing the valve for a suffi.cient time to ensure
complete discharge. Wait for 5 s before removing the
Table 2.9.18.-3. - Dimensions(l) ofjet tube with impaction plate Qin X Po

of apparatus C Qou t = Po - t:,.p
Type Code") Stage 1 Stage 2 Stage 3 Stage 4 Filter
(stage5 Po atmospheric pressure,
Distance 1 9.5 5.5 4.0 6.0 n.a. !:.P pressure drop over the meter.
(-.0+.5) (-.0+.5) (-.0+.5) (-.0+.5)
o Adjust the fiow control valve to achieve steady fiow through
Distance
Distance
2
3
26
8
31
5
33
5
30.5
5 5
the system at the required rate, º"w
( ± 5 per cent) . Switch
off the pump. Ensure that critical fiow occurs in the fiow
Distance 4 3 3 3 3 n.a. control valve by the following procedure.
Distance 5 o 3 3 3 3
Distance 6 (3) 20 25 25 25 25
E
Distance 7 n.a. n.a. n.a. 8.5 n.a.
Diameter e 25 14 8.0 21 14
(± .1)
Diameter d 50 30 20 30 n.a. o P3 P2 Impactor
Diameter e 27.9 16.5 10.5 23.9 n.a.

Vacuum
pump
Diameter f 31.75 22 14 31 22
(-.0+.5) Flow
r
g Control
Diameter 25.4 21 13 30 21 Valve
Diameter h n.a. n.a. n.a. 2.70 n.a.
(± .5) F VacuumTubing
Diameter j n.a. n.a. n.a. 6.3 n.a.
Connector
Diameter k n.a. n.a. n.a. 12.6 n.a. A B

Radius'4I r 16 22 27 28.5 O Figure 2.9.18.-8. - Experimental set-up for testing powder
Radius s 46 46 46 46 n.a. inhalers
Radius t n.a. 50 50 50 50
Angle w 10° 53° 53 ° 53° 53°
Table 2.9. 18.-4. - Component specification for Figure 2.9.18.-8
Angle u n.a. n.a. n.a. 45° n.a.
Code lIem Description
Angle v n.a. n.a. n.a. 60 ° n.a.
A Connector ID " 8 mm, e.g., short metal coupling, with
low-diameter branch to P3.
(1) Measures in millimetres with tolerances according to ISO 2768-m
unless otherwise stated B Vacuum tubing A length of suitable tubing having an ID " 8 mm
(2) Refer to Figure 2.9.18.-5 and an internal volume of 25 ± 5 mL.
(3) Including gasket C 2-way solenoid A 2-way, 2-port solenoid valve having a minimum
valve airnow resistan ce orifice with ID " 8 mm and
(4) Relative centreline of stage compartmen t an opening time '; 100 ms. (e.g. type 256-A08,
n.a. = not applicable Bürkert GmbH, D-74653 Ingelfingen), or
equivalen!.
D Vacuum pump Pump must be capable of drawing the required
f10w rate through the assembled apparatus
with the powder inhaler in the mouthpiece
081 adapter (e.g. product type 1023, 1423 or 2565,
Gast Manufacturing Inc., Benton Harbor, MI
060 49022), or equivalen!. Connect the pump to the
2-way solenoid valve using short and/ or wide
(ID ;, 10 mm) vacuum tubing and connectors to
minimise pump capacity requirements.
E Timer Timer capable to drive the 2-way solenoid
valve for the required duration (e.g. type
G814, RS Components International, Corby,
NN17 9RS U¡{), or equivalen!.
P2P3 Pressure Determine under steady·state now condition
measurements with an absolute pressure transducer.
F Flow control Adjustable regulating valve with maximum
valve C,;' 1, (e.g. type 8FV12LNSS, Parker Hannifin
plc. Barnstaple EX31 1NP UK), or equivalen!.
With the inhaler in place and the test fiow rate established,
measure the absolute pressure on both sides of the control
Figure 2.9.18.-6. - Apparatus C: details ofthe filter stage valve (pressure reading points P2 and P3 in Figure
(stage 5). Numbers refer to dimensions (0 = diameter). 2.9. 18. -8). A ratio P3/P2 ofless than or equal to 0.5
Uppercase letters refer to Table 2.9.18.-2. indicates critical fiow. Switch to a more powerful pump and
Dimensions in millimetres unless otherwise stated re-measure the test fiow rate if critical fiow is not indicated.
Dispense 20 mL of a solvent, capable of dissolving the active
Connect a fiowmeter to the induction port. Use a fiowmeter substance iTIto each of the 4 upper stages of the apparatus
calibrated for the volumetric fiow leaving the meter, or and replace the stoppers. Tilt the apparatus to wet the
calculate the volumetric fiow leaving the meter (Qour) using stoppers, thereby neutralising electrostatic charge. Place a
the ideal gas law. For a meter calibrated for the entering suitable mouthpiece adapter in position at the end of the
volumetric fiow (Qin) , use the following expression: induction port.
Drill, counter-bore and tap

tor an M-4 cap screw.
19.0
Note: use minimum clearance
for screw in the lower par! to aid
97
in precise alignment.
79
c.u
oo
1+
......
o
14.3 ~ 4-: 45 degree o
0*"
Q)<::>
31 .75~~g
Do not break edge
Joint must be leak tight
~
M-4 socket
head cap screw
Isometric view of induction port

1. Material may be aluminium, stainless steel or other suitable material.
2. Machine from 38 mm bar stock.
3. Bore 19 mm hole through bar.
4. Cut tube to exact 45 o as shown.
5. The inner bores and tapers should be smooth - surface roughness Ra approx. 0.4 jJm.
6. Mili joining cads of stock to provide a liquid tight leak·free seal.
7. Set up a holding fixture for aligning the inner 19 mm bore and for drilling and tapping M4 x 0.7 threads. There must be virtually no mismatch of the inner
bores in the miter joint.
Figure 2.9.18.-7. - Induction port
Dimensions in millimetres unless otherwise stated
Prepare the powder inhaler for use according to patient inner walls and the collection plate of each of the 4 upper
instructions. With the pump running and the 2-way solenoid stages of the apparatus into the solution in the respective
valve c1osed, locate the mouthpiece of the inhaler in the stage by carefully tilting and rotating the apparatus, observing
mouthpiece adapter. Discharge the powder into the that no liquid transfer occurs between the stages.
apparatus by opening the valve for the required time, Using a suitable method of analysis, determine the amount of
T (± 5 per cent) . Repeat the procedure. The number of active substance contained in each of the aliquots of solvent.
discharges should be minimised and typically would not be
Calculate the fine panic1e dose (see Calculations).
greater than lO. The number of discharges is sufficient to
ensure an accurate and precise determination of fine partic1e Apparatus D - Andersen cascade impactor
dose.
The Andersen 1 ACFM non-viable cascade impactor consists
Dismantle the filter stage of the apparatus. Carefully remove of 8 stages together with a final filter. Material of
the filter and extract the active substance into an aliquot of construction may be aluminium, stainless steel or other
the solvent. Remove the induction pon and mouthpiece suitable material. The stages are c1amped together and sealed
adapter from the apparatus and extract the active substance with O-rings. Critical dimensions applied by the
into an aliquot of the solvent. If necessary, rinse the inside of manufacturer of apparatus D are provided in
the inlet jet tube to stage 1 with solvent, allowing the solvent Table 2.9.18.-5. In use, sorne occ1usion and wear ofholes
to flow into the stage. Extract the active substance from the will occur. In-use mensuration tolerances need to be justified.
Figure 2.9.18.-9. - Apparatus D: Andersen cascade impactor used for pressurised inhalers
In the configuration used for pressurised inhalers (Figure respirable powder. It is connected to the induction port as
2.9.18.-9) the entry cone ofthe impactor is connected to an shown in Figure 2.9.18.-10. To accommodate high flow rates
induction port (see Figure 2.9.18.-7). A suitable mouthpiece through the impactor, the outlet nipple, used to connect the
adapter is used to provide an airtight seal between the inhaler impactor to the vacuum system is enlarged to have an
and the induction port. The front face of the inhaler intemal diameter of greater than or equal to 8 mm.
mouthpiece must be flush with the front face of the
induction porto
In the configuration for powder inhalers, a pre-separator is
placed aboye the top stage to collect large masses of non-
44 :n?1
38.3+ 0.2 .
19 --o ~ Cross-section
15 ---.J
14.4 ----J R12 .7 --.!
13 . R15
8~
---¡ "Jt<--- R15. 87~?05
, ..../1+1--- R14.4
Top view Groove for O-ring
R12 .7 R19
R44
R15.87 ~?05
R38.3 j¡.2
R6.70±0.03 10° ± 1° 107
Do not break edge / 106
45°± 3 °V = =,-- 102
~ 100
'" 94 Material: aluminium, stainless steel
or other suitable material.
Side view
Except where noted, all edges to be broken
and burrs removed.
Surface to be clean machine - tooled finish .
Interior bore surface roughness Ra
approx. 0.4 J.lm.
_ _ _ __ O O-ring: nominal dimensions: ID 29 mm,
" OD 32 mm, width 1.8 mm.
"'-- Do not break edge
Figure 2.9.18.-10. - Connection of the induction port to the preseparator of the Andersen cascade impactor
Dimensions in millimetres unless otherwise stated
Table 2.9.18.·5. - Critical dimensions for apparatus D as measured at the inlet to the induction port, to 28 .3 Umin
Description Number Dimension (mm) (± 5 per cent). Switch off the pump.
Stage Onozzle diameter 96 2.55 ± 0.025 Unless otherwise prescribed in the patient instructions, shake
the inhaler for 5 s and discharge one delivery to waste.
Stage 1 nozzle diameter 96 1.89 ± 0.025
Switch on the pump to the apparatus, locate the mouthpiece
Stage 2 nozzle diameter 400 0.914 ± 0.0127 end of the actuator in the adapter and discharge the inverted
Stage 3 nozzle diameter 400 0.711 ± 0.0127 inhaler into the apparatus, depressing the valve for a
sufficient time to ensure complete discharge. Wait for 5 s
before removing the assembled inhaler from the adapter.
Stage 5 nozzle diameter 400 0.343 ± 0.0127 Repeat the procedure. The number of discharges should be
Stage 6 nozzle diameter 400 0.254 ± 0.0127 mini mi sed and typically would not be greater than 10.
The number of discharges is sufficient to ensure an accurate
and precise determination of the fine particle dose. After the
final discharge, wait for 5 s and then switch off the pump.
Procedure lor pressurised inhalers Dismantle the apparatus. Carefully remove the filter and
extract the active substance into an aliquot of the solvent.
Assemble the Andersen impactor with a suitable filter in
Remove the induction port and mouthpiece adapter from the
place. Ensure that the system is airtight. In that respect,
apparatus and extract the active substance into an aliquot of
follow the manufacturer's instructions. Place a suitable
the solventoExtract the active substance from the inner walls
mouthpiece adapter in position at the end of the induction
and the collection plate of each of the stages of the apparatus
port so that the mouthpiece end of the actuator, when
into aliquots of solvent.
inserted, lines up along the horizontal axis of the induction
port and the inhaler unit is positioned in the same orientation Using a suitable method of analysis, determine the quantity
as the intended use . Connect a suitable pump to the outlet of of active substance contained in each of the aliquots of
the apparatus and adjust the air flow through the apparatus, solvent.
Calculate the fine particle dose (see Calculations). and 6.5 ~m. The collection efficiency curves for each stage
Procedure ¡or powder inhalers are sharp and minimise overlap between stages.
The aerodynamic cut-off diameters of the individual stages of this Material of construction may be aluminium, stainless steel or
apparatus are currently not well-established at fiow rates other other suitable material.
than 28.3 L/min. Users must justifY and validate the use of The impactor configuration has removable impaction cups
the impactor in the chosen conditions, when fiow rates with all the cups in one plane (Figures 2.9.18.-11/14). There
different from 28.3 Umin are selected. are 3 main sections to the impactor; the bottom frame that
Assemble the Andersen impactor with the pre-separator and holds the impaction cups, the seal body that holds the jets
a suitable filter in place and ensure that the system is airtight. and the lid that contains the interstage passageways (Figures
Depending on the product characteristics, the pre-separator 2.9.18.-11112). Multiple nozzles are used at all but the first
maybe omitted, where justified and authorised. Stages 6 and stage (Figure 2.9.18.-13). The fiow passes through the
7 may also be omitted at high fiow rates, if justified. The pre- impactor in a saw-tooth pattern.
separator may be coated in the same way as the plates or Critical dimensions are provided in Table 2.9.18.-6.
may contain 10 mL of a suitable solvent. Connect the In routine operation, the seal body and lid are held together
apparatus to a fiow system according to the scheme specified as a single assembly. The impaction cups are accessible when
in Figure 2.9.18.-8 and Table 2.9.18.-4. this assembly is opened at the end of an inhaler test.
Unless otherwise defined, conduct the test at the fiow rate, The cups are held in a support tray, so that all cups can be
Q,Utl used in the test for uniformity of delivered dose drawing removed from the impactor simultaneously by lifting out the
4 L of air from the mouthpiece of the inhaler and through tray.
the apparatus. An induction port with internal dimensions (relevant to the
Connect a fiowmeter to the induction port. Use a fiowmeter airfl.ow path) defined in Figure 2.9.18.-7 connects to the
calibrated for the volumetric fiow leaving the meter, or impactor inlet. A pre-separator can be added when required,
calculate the volumetric fiow leaving the meter (Qour) using typically with powder inhalers, and connects between the
the ideal gas law. For a meter calibrated for the entering induction port and the impactor. A suitable mouthpiece
volumetric fiow (Qin), use the following expression: adapter is used to provide an airtight seal between the inhaler
and the induction port.
_ Qin X Po Apparatus E contains a terminal Micro-Orifice Collector
Q out Po - f:,.P
- (MOC) that for most formulations will eliminate the need for
a final filter as determined by method validation. The MOC
Po = atmospheric pressure, is an impactor plate with nominally 4032 holes, each
t1P = pressure drop over the meter. approximately 70 ~m in diameter. Most particles not
captured on stage 7 of the impactor will be captured on the
Adjust the fiow control valve to achieve steady fiow through cup surface below the MOC. For impactors operated at 60
the system at the required rate, Q,ut (± 5 per cent). Ensure Umin, the MOC is capable of collecting 80 per cent of
that critical fiow occurs in the fiow control valve by the 0.14 ~m particles. For formulations with a significant fraction
procedure described for Apparatus C. Switch off the pump. of particles not captured by the MOC, there is an optional
Prepare the powder inhaler for use according to the patient filter holder that can replace the MOC or be placed
instructions. With the pump running and the 2-way solenoid downstream of the MOC (a glass fibre filter is suitable).
valve closed, locate the mouthpiece of the inhaler in the
Pro ce dure ¡or pressurised inhalers
mouthpiece adapter. Discharge the powder into the
apparatus by opening the valve for the required time, Place. cups into the apertures in the cup tray. Insert the cup
T (± 5 per cent). Repeat the discharge sequence. tray into the bottom frame, and lower into place. Close the
The number of discharges should be minimised and typically impactor lid with the seal body attached and operate the
would nN be greater than 10. The number of discharges is handle to lock the impactor together so that the system is
sufficient to ensure an accurate and precise determination of airtight.
fine particle dose. Connect an induction pon with internal dimensions defined
Dismantle the apparatus. Carefully remove the filter and in Figure 2.9.18.-7 to the impactor inlet. Place a suitable
extract the active substance into an aliquot of the solvent. mouthpiece adapter in position at the end of the induction
Remove the pre-separator, induction port and mouthpiece port so that the mouthpiece enq of the actuator, when
adapter from the apparatus and extract the active substance inserted, lines up along the horizontal axis of the induction
into an aliquot of the solvento Extract the active substance port. The front face of the inhaler mouthpiece must be fiush
from the inner walls and the collection plate of each of the with the front face of the induction port. When attached ro
stages of the apparatus into aliquots of solvent. the mouthpiece adapter, the inhaler is positioned in the same
orientation as intended for use. Connect a suitable pump to
Using a suitable method of analysis, determine the quantity
the outlet of the apparatus and adjust the air fiow through
of active substance contained in each of the aliquots of
the apparatus, as measured at the inlet to the induction port,
solvent.
to 30 Umin (± 5 per cent). Switch off the pump.
Calculate the fine particle dose (see Calculations).
Unless otherwise prescribed in the patient instructions, shake
Apparatus E the inhaler for 5 s and discharge 1 delivery to waste. Switch
on the pump to the apparatus. Prepare the inhaler for use
Apparatus E is a cascade impactor with 7 stages and a micro- according to the patient instructions, locate the mouthpiece
orifice collector (MOC). Over the fiow rate range of 30 end of the actuator in the adapter and discharge the inhaler
Umin to 100 Umin the 50 per cent-efficiency cut-off into the apparatus, depressing the valve for a sufficient time
diameters (D so values) range between 0.24 ~m to 11. 7 ~m, to ensure a complete discharge. Wait for 5 s before removing
evenly spaced on a logarithmic scale. In this fiow range, there the assembled inhaler from the adapter. Repeat the
are always at least 5 stages with Dso values between 0.5 ~lm
Induction por!
Pre-separator
Impactor body
Airflowoutlet
Clamping mechanism
Figure 2.9.18.-11. - Apparatus E (shown with the pre-separator in place)
Stage 1 nozzle Interstage passageway
Micro-orifice
collector (MOC)
Removable
+- Lid with seal

body attached
Location pin
- - - - - Location pin recess
Boltom trame with
Figure 2.9.18.-12. - Apparatus E showing component parts
procedure. The number of discharges should be minimised, from the apparatus and recover me deposited active
and typicalIy would not be greater man 10. The number of substance into an aliquot of solvento Open the impactor by
discharges is sufficient to ensure an accurate and precise releasing the handle and lifting me lid. Remove me cup tray,
determination of me fine partic1e dose. After me final with me colIection cups, and recover me active substance in
discharge, wait for 5 s and men switch off me pump. each cup into an aliquot of solvento
Dismantle me apparatus and recover the active substance as
folIows: remove me induction pOr! and moumpiece adapter
Table 2.9.18.-6. - Critical dimensions for apparatus E drawing 4 L of air from the mouthpiece of the inhaler and
Description Dimension through the apparatus. Connect a fiowmeter to the induction
(mm) port. Use a fiowmeter calibrated for the volumetric fiow
Pre-separator (dimension a- see Figure 2.9.18.-15) 12.8 ± 0.05 leaving the meter, or calculate the volumetric fiow leaving the
Stage 1* Nozzle diameter 14.3 ± 0.05 meter (Q,u,) using the ideal gas law. For a meter calibrated
for the entering volumetric fiow (Qin), use the following
Stage 2* Nozzle diameter 4.88 ± 0.04 expression:
Stage 3* Nozzle diameter 2.185 ± 0.02
Stage 4* Nozzle diameter 1.207 ± 0.01 Qin X Po

Qout = Po - D..P
Po atmospheric pressure,
Stage 7* Nozzle diameter 0.206 ± 0.01 I!J.P pressure drop over the meter.
MOC* approx. 0.070 Adjust the fiow control valve to achieve steady fiow through
Cup depth (dimension b - see Figure 2.9.18.-14) 14.625 ± 0.10 the system at the required rate, Qout (± 5 per cent). Ensure
that critical fiow occurs in the fiow control valve by the
Collection cup surface roughness (Ra) 0.5 - 2 ¡¡m
procedure described for Apparatus C. Switch off the pump.
Stage 1 nozzle to seal body distance** - dimension c O± 1.18
Prepare the powder inhaler for use according to the patient
Stage 2 nozzle to seal body distance** - dimension c 5.236 ± 0.736 instructions. With the pump running and the 2-way solenoid
Stage 3 nozzle to seal body distance** - dimension c 8.445 ± ü.41O valve closed, locate the mouthpiece of the inhaler in the
mouthpiece adapter. Discharge the powder into the
Stage 4 nozzle to seal body distance** - dimension c 11.379 ± 0.237
apparatus by opening the valve for the required time,
Stage 5 nozzle to seal body distance** - dimension c 13.176 ± 0.341 T (± 5 per cent). Repeat the discharge sequence.
Stage 6 nozzle to seal body distance** - dimension c 13.999 ± 0.071 The number of discharges should be minimised and typically
would not be greater than 10. The number of discharges is
Stage 7 nozzle to seal body distance** - dimension c 14.000 ± 0.071 sufficient to ensure an accurate and precise determination of
MOC nozzle to seal body distance** - dimension c 14.429 to 14.571 fine particle dose.
* See Figure 2.9.18_-13 Dismantle the apparatus and recover the active substance as
*. See Figure 2.9.18.-14 follows: remove the induction port and mouthpiece adapter
from the pre-separator, when used, and recover the deposited
active substance into an aliquot of solventoWhen used,
Using a suitable method of analysis, determine the quantity
remove the pre-separator from the impactor, being careful to
of active substance contained in each of the aliquots of
avoid spilling the cup liquid into the impactor. Recover the
solvent.
active substance from the pre-separator.
Calculate the fine partic1e dose (see Calculations).
Open the impactor by releasing the handle and lifting the lid.
Procedure lor powder inhalers Remove the cup tray, with the collection cups, and recover
Assemble the apparatus with the pre-separator (Figure the active substance in each cup into an aliquot of solvento
2.9 .18.-15) . Depending on the product characteristics, the Using a suitable method of analysis, determine the quantity
pre-separator may be omitted, where justified. of active substance contained in each of the aliquots of
Place cups into the apertures in the cup tray. Insert the cup solvento
tray into the bottom frame, and lower into place. Close the Calculate the fine partic1e dose (see Calculations).
impactor lid with the seal body attached and operate the
handle to lock the impactor together so that the system is Calculations
airtight. From the analysis of the solutions, calcula te the mas s of
When used, the pre-separator should be assembled as active substance deposited on each stage per discharge and
follows: assemble the pre-separator insert into the pre- the mass of active substance per discharge deposited in the
separator base. Fit the pre-separator base to the impactor induction port, mouthpiece adapter and when used, the pre-
inlet. Add 15 mL of the solvent used for sample recovery to separator.
the central cup of the pre-separator inserto Place the pre- Starting at the final collection site (filter or MOC), derive a
separator body on top of this assembly and close the 2 table of cumulative mas s versus cut-off diameter of the
catches. respective stage (see Tables 2.9.18 .-7 for Apparatus C,
Connect an induction port with internal dimensions defined 2.9.18.-8 for Apparatus D, 2.9.18 .-9 for Apparatus E) .
in Figure 2.9.18.-7 to the impactor inlet or pre-separator Calculate by interpolation the mass of the active substance
inlet. Place a suitable mouthpiece adapter in position at the less than 5 ¡.tm. This is the Fine Partic1e Dose (FPD).
end of the induction port so that the mouthpiece end of the If necessary, and where appropriate (e.g., where there is a
inhaler, when inserted, lines up along the horizontal axis of log-normal distribution), plot the cumulative fraction of
the induction port. The front face of the inhaler mouthpiece active substance versus cut-off diameter (se e Tables
must be fiush with the front face of the induction port. When 2.9 .18.-7/9) on log probability paper, and use this plot to
attached to the mouthpiece adapter, the inhaler is positioned determine values for the Mass Median Aerodynamic
in the same orientation as intended for use. Connect the Diameter (MMAD) and Geometric Standard Deviation
apparatus to a fiow system according to the scheme specified (GSD) as appropriate. Appropriate computational methods
in Figure 2.9 .18.-8 and Table 2.9.18.-4. may also be used.
Unless otherwise prescribed, conduct the test at the fiow
rate, Q,,,,, used in the test for uniformity of delivered dose
V -A3 72 Appendix XII e 2014
Stage 2 Stage 4 Stage 6 MOC

6 holes 52 holes 396 holes 4032 holes
Stage 1 Stage 3 Stage 5 Stage 7

1 hole 24 holes 152 holes 630 holes
Figure 2.9.18.-13. - Apparatus E: nozzle configuration
Interstage passage Interstage passage

( lo next slage ( from previous stage
/ 1 I
,-- Lid
t t
f 1 f J
,-- Seal body
~
l eL
~
J ,-- Cup tray
~ Air flow r(
~~D¡O¡O¡qo¡o¡7
Cup tray b ./Bottom
, frame
.)
Collection cup ~ Multi-nozzle stage /
Figure 2.9.18.-14. - Apparatus E: configuration of interstage passageways
Pre-separator body
/
/
Catch
- ___ Nozzle diameter,
dimension a
Central cup
t
o = o
t
Pre-separator insert
Figure 2.9.18.-15. - Apparatus E : pre-separator configuration

TabIe 2.9.18.-7. - Calculations for Apparatus C. Use q = vI(60/ Q), where Q is the test flow rate in litres per minute
(Qout for powder inhalers)
Cut-oCC diameter Mass oC active substance deposited Cumulative mass oC active substance Cumulative fraction oC active substance
(um) per discharge deposited per discharge (per cent)
d, = 1.7 x q mass fram stage 5, m5' C4 - ms f, = (e';e) x 100
d3 = 3.1 x q mass fram stage 4, m. c¡ = e. + m. f, = (c/c) x 100
d, = 6.8 x q mass fram stage 3, m, c,=e,+m, f, = (c,/e) x 100
mass fram stage 2, m, c=c2 +m Z 100

• Stage 5 is the filter stage
TabIe 2.9.18.-8. - Calculations for Apparatus D when used at a flow rate of28.3 L/ min
Cut-oCC diameter Mass of active substance deposited Cumulative mass aC active substance Cumulative fractian of active substance
(um) per discharge deposited per discharge (per cent)
d, = 0.4 mas s fram stage 8, m, c7 - ms f, = (c.,/ e) x 100
d, = 0.7 mass fram stage 7, m, c6 =c7 +m 7 f, = (c.;e) x 100
d5 = 1.1 mass fram stage 6, m, cS =c6 +mij f5 = (e';e) x 100
d, = 2.1 mass fram stage 5, m, e4 = es + ms f. = (e';e) x 100
d, = 3.3 mass fram stage 4, m. e3 = c4 + m4 f, = (e,le) x 100
d, = 4.7 mass fram stage 3, m, cZ =c)+m 3 f, = (c,/e) x 100
d, = 5.8 mass fram stage 2, m, el=c,+m, f, = (c/ c) x 100
do = 9.0 mass fram stage 1. m, Co = el + mI fo = (eole) x 100
mass fram stage O, mo c=co +mo 100
TabIe 2.9.18.-9. - Calculations for Apparatus E. Use q = (60/Q1, where Q is the test flow rate in litres per minute, and x
is listed in the table
Cut-oCf diameter x Mass of active substance Cumulative mass oC active substance Cumulative fraction of active
(um) deposited per discharl!e deposited per discharl!e substance (per cent)
d, = 0.34 x q 0.67 mass fram MOC ar terminal c., - m, F, = (c.,/ e) x 100
filter, m,
d, = 0.55 x q 0.60 mass from stage 7, m, <;¡ = c., + m, F, = (c.;e) x 100
d, = 0.94 x q 0.53 mass fram stage 6, m, c,=<;¡+m, F, = (c,/e) x 100
d. = 1.66 x q 0.47 mass fram stage 5, m, c4 =cS +m S F. = (e';e) x 100
d, = 2.82 x q 0.50 mass fram stage 4, m. c¡=e.+m, F, = (e,le) x 100
d, = 4.46 x q 0.52 mass fram stage 3, m, c2 =c3 +m 3 F, = (e,!e) x 100
dI = 8.06 x q 0.54 mass fram stage 2, m, el = C, + m, F, = (c/ c) x 100
mass from stage 1. mI e = el + mi 100
8. Preparations for Nebulisation: Characterisation ACTIVE SUBSTANCE DELIVERY RATE ANO

(Ph. Eur. methad 2.9.44) TOTAL ACTIVE SUBSTANCE DELIVERED
Products used for nebulisation and intended for pulmonary These tests are performed to assess the rate of delivery to the
delivery are characterised using the following tests: patient and the total active substance delivered to the patient,
- - Active substance delivery rate and total active using standardised conditions of volumetric fiow rateo It is
substance delivered; essential that breath-enhanced and breath-actuated nebulisers
be evaluated by a breathing simulator, as the output of these
- - Aerodynamic assessment of nebulised aerosols .
types of device is highly dependent on inhalation fiow rateo
These tests standardise the approach given to the assessment The methodology below describes the use of a standard
of the dose that would be delivered to a patient but are not breathing pattem defined for adults. Should a particular
intended to provide assessment of the nebuliser device itself, product for nebulisation only be indicated for paediatric
which is described in the European standard EN (i.e. neonate, infant or child) use, then paediatric breathing
13544-1 :2007+Al :2009, Respiratory therapy equipment - pattem(s) must be used. Breathing pattems are used, rather
Part 1: N ebulizing systems and their components. than continuous fiow rates, to provide a more appropriate
The mass- rather than the number-weighted size distribution measure of the mass of active substance that would be
is more appropriate to evaluate product performance. Indeed, delivered to patients.
active substance mass as a function of aerodynamic diameter Active substance delivery rate and total active substance
is more indicative of therapeutic effect within the respiratory delivered are appropriate characteristics because they allow
tracto
the mass delivered to be characterised in a standard way soaked with the preparation, this time can be decreased.
regardless of the nebuliser used. Accordingly, the test At the end of this initial period, stop the nebuliser.
methodology described below allows that the mas s of active Place a fresh filter and filter holder in position and continue
substance delivered in the 1sr period (typically 1 min) is until nebulisation ceases. Interrupt nebulisation and exchange
measured (consequently giving an assessment of active filters if necessary, to avoid filter saturation.
substance delivery rate) as well as capturing the total mass of
active substance delivered. Results
Using a suitable method of analysis, determine the mas s of
Apparatus active substance collected on the filters and filter holders
Breathing simulator A commercially available breathing during each time interval. Determine the active substance
simulator, which is able to generate the breathing profiles delivery rate by dividing the mass of active substance
specified in Table 2.9 .44.-1 , is used for the test. collected on the first inhalation filter by the time interval
The breathing pro file indicated for adults is used unless the used for collection. Determine the total mass of active
medicinal product is specifically intended for use in substance delivered by summing the mass of active substance
paediatrics, where alternate patterns should be used, as collected on all inhalation filters and filter holders.
indicated in Table 2.9.44.-1.
AERODYNAMIC ASSESSMENT OF NEBULISED
Table 2.9.44.-1. - Breathing simulator specifications AEROSOLS
Item Specification Nebulised products need to be size-characterised at fiow rates
lower than the range that is normally used for powder
Adult Neonate lnfant Child
inhalers and metered-dose inhalers. A fiow rate of 15 Umin
Tidal 500 mL 25 mL 50 mL 155 mL is recommended in the European standard because this value
volume
represents a good approximation to the mid-inhalation fiow
Frequency 15 40 30 25
cycles/ min cycles/ min cycles/ min cycles/ min
rate achievable by a tidally breathing healthy adult (500 mL
tidal volume) .
Waveform sinusoidal sinusoidal sinusoidal sinusoidal
Although low-angle laser light scattering instruments (laser
Inhala· 1:1 1:3 1:3 1:2
tion/exha- diffractometers) can provide rapid size-distribution
lation ratio measurements of nebuliser-generated aerosols, these
techniques do not detect the active substance; rather they
Filter system A suitably validated low-resistance filter, measure the size distribution of the droplets irrespective of
capable of quantitatively collecting the aerosol and enabling their contento This may not be a problem with homogeneous
recovery of the active substance with an appropriate solvent, solutions, but can result in significant error if the product to
is used for the test. The dead volume of the filter casing be nebulised is a suspension, or if droplet evaporation is
does not exceed 10 per cent of the tidal volume used in the significant as can be the case with certain nebuliser types.
breath simulation. Cascade impactors enable the aerosol to be characterised
unambiguously in terms of the mas s of active substance as a
Method function of aerodynamic diameter. Laser diffraction may be
Attach the filter (contained in the filter holder) (A) to the used if validated against a cascade impaction method.
breath simulator (B) according to Figure 2.9.44.-1. Fill the Apparatus E (see below under Apparatus), a cascade
nebuliser (C) with the volume of the medicinal product as impactor, has been calibrated at 15 Umin specifically to
specified in the patient instructions. Attach the mouthpiece meet the recommendation of the European standard, and is
of the nebuliser to the inhalation filter using a mouthpiece therefore used for this test. Determining mass balance in the
adapter if required, ensuring that connections are airtight. same way as for powder inhalers and metered-dose inhalers is
Make sure the nebuliser is positioned in the same orientation not straightforward, in that the dose is being captured as a
as intended for use; this may require tilting the breathing continuous output, and hence is not inc1uded. As part of
simulator and filter holder. Set the breathing simulator to method development, recovery experiments must be
genera te the specified breathing pattern. performed to validate the method.
It is also recognised that the control of evaporation of
droplets produced by nebulisers may be critical to avoid bias
in the droplet size assessment process. Evaporation can be
minimised by cooling the impactor to a temperature of about
5 oC, typically achieved by cooling the impactor in a
refrigerator for about 90 min. Typically, at least after each
A B day of use, the 'apparatus must be fully c1eaned, inc1uding the
A. inhalation filter and filter holder B. breathing simulator C. nebuliser inter-stage passageways, in view of the greater risk of
corrosion caused by the condensation/accumulation of saline-
Figure 2.9.44.-1. - Experimental set-up for breathing simulator
testing containing droplets on inter-stage metalwork associated with
cooling the impactor. It is recommended to dry all surfaces
of the apparatus after each test, for example with compres sed
Start the breathing simulator then, at the beginning of an air. Note: the micro-orifice collector (MOC) should not be
inhalation cyc1e, start the nebuliser. Operate the nebuliser for dried with compres sed air,
a defined initial time period. The time chosen, usually
60 ± 1 s, must allow sufficient active substance deposition Apparatus
on the inhalation filter to allow quantitative analysis. If the A detailed description of Apparatus E and the induction port
quantity of active substance deposited on the inhalation filter is contained in general chapter 2.9.18, and inc1udes details of
in 60 s is insufficient for this analysis, the length of the time critical dimensions and the qualification process for the
interval for aerosol collection can be increased. If the filter is impactor (stage mensuration).
e D
A. nebuliser B. induction port C. impactor (apparatus El D. f10w control valve E. vacuum so urce
Figure 2.9.44.-2. - Apparatus E for measurin,q the size distribution of preparations for nebulisation
A back-up filter in addition to the micro-orifice collector Connect a flow meter, calibrated for the volumetric flow
(MOC) must be used to ensure quantitative recovery of leaving the meter, to the induction port. Adjust the flow
active substance from the nebulised aerosol at the specified control valve located between the impactor and the vacuum
flow rate of 15 Umin. The filter is located below the MOC source to achieve a steady flow through the system at 15
(internal filter option) or a filter in holder, external to the Umin (± 5 per cent). Remove the flow meter.
impactor, is used to capture any fine droplets that pass Make sure the nebuliser is positioned in the same orientation
beyond the last size fractionating stage. as intended for use then attach the mouthpiece of the
A pre-separator is not used for testing nebuliser-generated nebuliser to the induction port, using a mouthpiece adapter if
aerosols. required, ensuring that connections are airtight. Switch on
Method validation the flow/compressor for the nebuliser. Sample for a
predetermined time (To). Once determined, this time (To)
Impactor stage overloading During method development
must be defined and used in the analytical method for a
and validation, it is important to confirm that the volume of
particular medicinal product to ensure that mass fraction
liquid sampled from the nebuliser does not overload the
data can be compared. At the end of the sampling period,
impactor. Visual inspection of the collection surfaces on
switch off the driving gas flow/compressor to the nebuliser,
stages collecting most of the droplets may reveal streaking if
remove the nebuliser from the induction port and switch off
overloading has occurred. This phenomenon is usually also
the flow from the vacuum source to the impactor.
associated with an increase in mass of active substance '
collected on the final stage and back-up filter. Reducing the Dismantle the impactor and, using a suitable method of
sampling period (To) is the most effective way to avoid analysis, determine the mass of active substance collected in
overloading in any given system, balancing overloading with the induction port, on each stage and on the back-up
analytical sensitivity. filter/external filter as described for Apparatus E in general
chapter 2.9.18. Add the mass of active substance collected in
Re-entrainment Droplet bounce and re-entrainment are
the MOC to that deposited on the back-up filter/external
less likely with nebuliser-produced droplets than with solid
filter and treat as a single sample for the purpose of
particles from inhalers and for that reason coating would not
subsequent calculations.
normally be required.
Calculate the mass fraction' ( Fm ,comp) of the active substance
Method deposited on each component of the impactor, commencing
Pre-cool the assembled impactor and induction port in a with the induction port and proceeding in order through the
refrigerator (set at about 5 OC) for not less than 90 min and impactor, using the following expression:
start the determination within about 5 min of removal of the
impactor from the refrigerator. Other methods that maintain m comp
the impactor at a constant temperature (for example, use of a Fm ,co mp = -----¡,;¡-
cooling cabinet) can also be employed when validated.
Set up the nebuliser with a supply of driving gas (usually air mass associated with the component under
or oxygen), or use a compressor, at the pressure and flow evaluation;
rate specified by the manufacturer of the nebuliser. Take M total mas s collected by the system .
precautions to ensure that the gas supply line does not Present Fm ,com p in order of location within the measurement
become detached from the nebuliser when under pressure. equipment, beginning at the induction port and ending with
Fill the nebliliser with the volume of the medicinal product the back-up filter of the impactor (see Figure 2.9 .44.-3) .
as specifi~d in the patient instructions .
Where appropriate, Fm,comp for adjacent stages of the
Remove the impactor from the refrigerator. Attach the impactor may be combined in order to report the mass
induction port to the impactor, and connect the outlet of the fraction collected on a group of stages as a single value.
impactor/external filter to a vacuum source that is capable of
drawing air through the system at 15 Umin as specified in
Figure 2.9.44.-2. Turn on the flow through the impactor.
0 . 4 , - - - - -- - - -- - - - - - --
2 alternatives is considered as evidence that the medicinal
product tested complies with general chapter 2.9.40. The
2 alternatives are considered equivalent in their
0. 3 demonstration of compliance with general chapter 2.9.40.
Alternative 1 (parametric)
Select not fewer than 100 units according to a predefined
sampling plan.
The consistency of dosage units is evaluated by content
0.1 uniformity or mass variation as prescribed in Table 2.9.40.-1.
Calculate the acceptance value (A V) using the following
expression:
t
o
Il.
Q.l
O>
N
(!)
O>
Q)
O>
IM-x l+ ks
e .!9 El
o <7l (IJ (IJ
~ for which the terms are defined in Table 2.9.40.-2, but using
:::J
U
E the sample size-dependent value for k defined in
Table 2.9.47.-1.
Figure 2.9.44.-3. - E'xample af mass fracfian of droplets
Criteria
presented in terms of loca tio n within the sampling slJstem
Apply the following criteria, unless otherwise specified.
Determine the cumulative mass-weighted particle-size The requirements for dosage form uniformity are met if:
distribution of the aerosol size-fractionated by the impactor 1. the acceptance value (A V) is less than or equal ro Ll;
in accordance with the procedure given in general chapter and
2.9.18. Starting at the filter, derive a cumulative mass versus 2. in the calculation of acceptance value (A V) under
effecüve cut-off diameter of the respective stages (see content uniformity or under mas s variation, the number
Table 2.9.44.-2 for the appropriate cut-off diameters at ofindividual dosage units outside (l ± L2 x O.OI )M is
15 Umin). Plot the cumulative fraction of active substance less than or equal ro e2 as defined for a given sample
versus cut-off diameter in a suitable format, for example size n in Table 2.9.47 .-1.
logarithmic or log-probability format. Where appropriate,
Unless otherwise specified, Ll is 15.0 and L2 is 25.0.
determine by interpolation the fraction either below a given
size or between an upper and a lower size limit. Table 2.9.47.-1. is ro be interpreted as follows:
- for a sample size of n = 400, enter the table at n 2: 385:
Table 2.9.44.-2 . - Cut-off sizes for Apparatus E al 15 L/m in k = 2.23 and e2 = 3;
Stage Cut-off diameter (¡1m) - for a sample size of n = 450, enter the table at 17 2: 407:
k = 2.24 and e2 = 3;
14.1
- for a sample size of 17 = 500, enter the table at 17 2: 490:
2 8.61 k= 2.24 and e2 = 4.
3 5.39
Alternative 2 (non-parametric)
4 3.30
Select not fewer than 100 units according to a predefined
5 2.08 sampling plan.
6 1.36 The consistency of dosage units is evaluated by content
uniformity or mas s variation as prescribed in Table 2.9.40.-1.
7 0.98
Assay individually or weigh the units and calculate individual
contents"as prescribed in general chapter 2. 9.40. Count the
If necessary, and where appropriate, determine values for the number of individual dosage units with a content outside (l
mas s median aerodynamic diameter (MMAD) and the ± L1 x O.OI)M and the number of individual dosage units
geometric standard deviation (GSD), as appropriate. with a content outside (l ± L2 x O.OI)M. Evaluate ifthe
values are within the Iimits defined in Table_2.9.47 .-2.
9. Demonstration ofUnifonnity ofDosage Units
Using Large Samples Sizes Criteria
(Ph. Eu/'. method 2.9.47) Apply the following criteria, unless otherwise specified.
The proeedure is intended for, but not limited to, the evaluation of The requirements for dosage form uniformity are met if:
medicinal produets that are manufactured using proeess analytical 1. the number of individual dosage units outside (1 ± L1
teehnology (PAT) methodology. x O.OI)M is less than or equal to el; and
Compliance with general chapter 2.9.40. Uniformity of dosage 2. the number of individual dosage units outside (1 ± L2
units can be demonstrated by the following procedure, when x O.OI)M is less than or equal ro e2.
large samples (sample size n 2: 100) are evaluated. el and e2 for a given sample size 11 are defined in
Application of this chapter does not constitute a mandatory Table 2.9.47.-2. Unless otherwise specified, L1 is 15.0 and
requirement. Ir presents 2 alternative tests (Alternative I and L2 is 25.0.
Alternative II). Fulfilling the requirements of either of the
Table 2.9.47.-1. - Acceptability constant (k) and acceptable number af dasage units
with a content autside (1 ± L2 x O.Ol)M (= c2) (ar a given sample size n
n ~) k e2 n~) k e2 n~) k e2 n~) k e2 n~) k e2 n~) k e2
100 2.15 804 2.26 2480 2.29 23 4366 2.30 41 6252 2.31 59 8243 2.31 78
7
105 2.16 905 2.27 2585 2.29 24 4471 2.30 42 6357 2.31 60 8347 2.31 79
908 2.27 8 2690 2.29 25 4576 2.30 43 6462 2.31 61

120 2.17 O 8452 2.31 80
1013 2.27 9 2794 2.29 26 4680 2.30 44 6566 2.31 62
139 2.18 8557 2.31 81
1118 2.27 10 2899 2.29 27 4785 2.30 45 6671 2.31 63
161 2.19
8662 2.31 82
1223 2.27 3004 2.29 28 4890 2.30 46 6776 2.31 64
176 2.19 11 8767 2.31 83
1276 2.28 3109 2.29 4995 2.30 47 6881 2.31 65
189 2.20 29
1 8871 2.31 84
1328 2.28 12 3171 2.30 5099 2.30 48 6985 2.31 66
224 2.21
1432 2.28 13 3213 2.30 30 5204 2.30 49 7090 2.31 67 8976 2.31 85
270 2.22
1537 2.28 14 3318 2.30 31 5309 2.30 50 7195 2.31 68 9081 2.31 86
280 2.22
2 1642 2.28 15 3423 2.30 32 5414 2.30 51 7300 2.31 69 9186 2.31 87
328 2.23
1747 2.28 16 3528 2.30 33 5519 2.30 52 7404 2.31 70
9290 2.3 1 88
385 2.23
1851 2.28 3633 2.30 34 5623 2.30 53 7509 2.31 71
3 9395 2.31 89
17
407 2.24
1918 2.29 3737 2.30 35 5728 2.30 54 7614 2.31 72
9500 2.31 90
490 2.24 5833 2.30 55 7719 2.31 73
1956 2.29 18 3842 2.30 36
4
9605 2.31 91
516 2.25 2061 2.29 19 3947 2.30 37 5938 2.30 56 7824 2.31 74
9710 2.31 92
594 2.25 2166 2.29 20 4052 2.30 38 6042 2.30 7928 2.31 75
5 57
672 2.26 8033 2.31 76 9814 2.31 93
2270 2.29 21 4156 2.30 39 6136 2.31
699 2.26 6 2375 2.29 22 4261 2.30 40 6147 2.31 58 8138 2.31 77 9919 2.31 94
2014
V-A378 Appendix XII e
Table 2.9.47.-2. - Acceptable number of individual dosage units with a con ten! ou!side (1 ± L1 x O.Ol)M (= el) and
(1 ± L2 x O.Ol)M (= c2) respectively, for a given sample size n
el e2 n(?) el e2 n (?) el e2 n (?) el e2 n (?) el e2 n (?) e1 e2 n(?) e1 e2

n(?)
I I
100 3 1432 35 2899 67 4366 98 5833 129 7300 160 8767 191
123 4 O 1476 36 13 2935 68 27 4377 99 41 5835 130 7304 161 8780 192 83
55 69
159 5 1521 37 2981 69 4424 100 5883 131 7351 162
8828 193
176 5 1537 37 3004 69 4471 101 5930 132 7399 163
8871 193
196 1566 38 14 3027 70 28 4518 102 42 5938 132 7404 163
6
1 8875 194
1611 39 3073 71 4565 103 5977 133 56 7447 164 70 84
234 7
8923 195
1642 39 3109 71 4576 103 6024 134 7494 165
273 8
1656 40 6042 134 7509 165 8971 196
280 8 3120 72 4612 104 43
15 29
1701 41 3166 73 6072 135 57 7542 166 71 8976 196
313 9 2 4658 105
1746 42 3212 74 6119 136 7589 167 9019 197 85
353 10 4680 105
1747 42 3213 74 6147 136 7614 167
385 10 4705 106 44 9066 198
1791 43 16 3259 75 30 6166 137 58 7637 168 72
394 11 4752 107 9081 198
3 1836 44 6214 138 7684 169
3305 76 107
434 12 4785 9114 199 86
1851 44 6252 138 7719 169
3318 76
476 13 4799 108 45 9162 200
1882 45 17 139 7732 170 73
3351 77 31 6261
490 13 4846 109 59
1927 46 9186 200
3398 78 6308 140 7779 171
517 14 4 4890 109
1956 46 9210 201 87
3423 78 6355 141 7824 171
559 15 4893 110
1972 47 18 46 9257 202
3444 79 32 6357 141 7827 172
594 15 4940 111 74
2018 48
3491 80 6403 142 60 7875 173 9290 202
601 16 2061 48 4987 112
5 3528 80 6450 143 7922 174 9305 203 88
644 17 2063 49 4995 112
19 3537 81 6462 143 7928 174
5034 113 47 9353 204
686 18 2109 50 33
3584 82 6498 144 61 7970 175 75
699 18 2154 51 5081 114 9395 204
3630 83 6545 145 8017 176
729 19 6 2166 51 5099 114 9401 205
3633 83 6566 145 8033 176 89
772 20 2200 52 20 5128 115 48 9449 206
3677 84 34 6592 146 62 8065 177 76
804 20 2246 53 5175 116
6640 147 8113 178 9496 207
3723 85
815 21 2270 53 5204 116
3737 85 .6671 147 8138 178 9500 207
7
22 2291 54 21 5222 117 49
858
3770 86 35 6687 148 63 8160 179 77 9544 208 90
2337 55 5269 118
-
902 23
3817 87 6734 149 8208 180 9592 209
2375 55 5309 118
908 23 180
3842 87 6776 149 8243
2383 56 9605 209
945 24 8 22 5317 119
3863 88 36 50 6782 150 8256 181 78
2429 57 64 9640 210 91
989 25 5364 120
3910 89 6829 151 8303 182
2475 58 9688 21 1
1013 25 5411 121
3947 89 6877 152 8347 182
2480 58
1033 26 9 5414 121 9710 211
3956 90 6881 152 8351 183
2520 59 23 37 79
1077 27 5458 122 51 9735 212 92
4003 91 6924 153 65 8399 184
2566 60 ,
1118 27 5505 123 6972 154 8446 185 9783 21 3
2585 60 4050 92
1121 28 5519 123 6985 154 8452 185
2612 61 24 4052 92 9814 21 3
10
1165 29 4097 93 38 5552 124 52 7019 155 66 8494 186 80
2658 62 9831 214 93
1209 30 4143 94 5599 125 7067 156 8542 187
2690 62 9879 215
1223 30 4156 94 5623 125 7090 156 8557 187
2704 63 25 9919 215
1253 31 11 4190 95 39 5647 126 53 711 4 157 67 8589 188 81
2750 64
9927 216
1298 32 2794 64 4237 96 5694 127 7161 158 8637 189
9975 217 94
1328 32 2796 65 4261 96 5728 127 7195 158 8662 189
26 10023 218
1342 33 12 2843 66 4284 97 40 5741 128 54 7209 159 68 8685 190 82
1387 34 2889 67 4330 98 5788 129 7256 160 8732 191 10070 219
Table 2.9.47 .-2 is to be interpreted as follows: - for a sample size of n = 500, enter the table at n 2: 490:
- for a sample size of n = 400, enter the table at n 2: 394: el = 13 and c2 = 4.
el = 11 and c2 = 3;
- for a sample size of n = 450, enter the table at n 2: 434:
el = 12 and c2 = 3;
2014 Appendix XIII A V-A379
determination is to be carried out.

Appendix XIII In order to check that the environment is suitable for the
test, that the glassware is properly c\eaned and that the water
to be used is partic\e-free, the following test is carried out:
Particulate Contamination determine the particulate contamination of 5 samples of
A. Particulate Contamination: particle-free water R, each of 5 mL, according to the method
Sub-visible Particles 1 described below. If the number of partic\es of 10 fim or
greater size exceeds 25 for the combined 25 mL, the
(Ph. Eur. method 2.9.19) precautions taken for the test are not sufficient.
Particulate contamination of injections and infusions consists The preparatory steps must be repeated until the
of extraneous, mobile undissolved partic\es, other than gas environment, glassware and water are suitable for the test.
bubbles, unintentionally present in the solutions. METHOD
For the determination of particulate contamination 2 Mix the contents of the sample by slowly inverting the
procedures, Method 1 (Light Obscuration Partic\e Count container 20 times successively. If necessary, cautiously
Test) and Method 2 (Microscopic Partic\e Count Test), are remove the sealing c\osure. Clean the outer surfaces of the
specified hereinafter. When examining injections and container opening using a jet of particle-free water R and
infusions for sub-visible partic\es, Method 1 is preferably remove the c\osure, avoiding any contamination of the
applied. However, it may be necessary to test sorne contents. Eliminate gas bubbles by appropriate mea sures
preparations by the light obscuration partic\e count test such as allowing to stand for 2 min or sonicating.
followed by the. microscopic partic\e count test to reach a For large-volume parenterals, single units are tested.
conc\usion on conformance to the requirements. For small-volume parenterals less than 25 mL in volume, the
Not all parenteral preparations can be examined for sub- contents of lOor more units are combined in a c\eaned
visible partic\es by one or both of these methods. When container to obtain a volume of not less than 25 mL; where
Method 1 is not applicable, e.g. in case of preparations justified and authorised, the test solution may be prepared by
having reduced c\arity or increased viscosity, the test is mixing the contents of a suitable number of vials and diluting
carried out according to Method 2. Emulsions, colloids, and to 25 mL with particle-free water R or with an appropriate
liposomal preparations are examples. Similarly, products that solvent without contamination of partic\es when particle-free
produce air or gas bubbles when drawn into the sensor may water R is not suitable. Small-volume parenterals having a
also require microscopic partic\e count testing. If the viscosity volume of 25 mL or more may be tested individually.
of the preparation to be tested is sufficiently high so as to Powders for parenteral administration are reconstituted with
prec\ude its examination by either test method, a quantitative particle-free water R or with an appropriate solvent without
dilution with an appropriate diluent may be made to decrease contamination of partic\es when particle-free water R is not
viscosity, as necessary, to allow the analysis to be performed. suitable.
The results obtained in examining a discrete unit or group of The number of test specimens must be adequate to provide a
units for particulate contamination cannot be extrapolated statistically sound assessment. For large-volume parenterals
with certainty to other units that remain untested. Thus, or for small-volume parenterals having a volume of 25 mL or
statistically sound sampling plans must be developed if valid more, fewer than 10 units may be tested, based on an
inferences are to be drawn from observed data to characterise appropriate sampling plan.
the level of particulate contamination in a large group of
Remove 4 portions, each of not les s than 5 mL, and count
units.
the number of partic\es equal to or greater than 10 fim and
Method 1. Light obscuration partic1e count test 25 fim. Disregard the result obtained for the first portion,
and calculate the mean number of partic\es for the
Use a suitable apparatus based on the principie of light preparation to be examined.
blockage which allows an automatic determination of the size
of partic\es and the number of partic\es according to size. EVALUATION
For preparations supplied in containers with a nominal
The apparatus is calibrated using suitable certified reference
volume of more than 100 mL, apply the criteria of test 1.A.
materials consisting of dispersions of spherical partic\es of
known sizes between 10 ~Lm and 25 fim. These standard For preparations supplied in containers with a nominal
partic\es are dispersed in particle-free water R. Care must be volume of less than 100 mL, apply the criteria of test l.B.
taken to avoid aggregation of partic\es during dispersion. For preparations supplied in containers with a nominal
GENERAL PRECAUTIONS volume of 100 mL, apply the criteria of test l.B.
The test is <;arried out under conditions limiting particulate If the average number of partic\es exceeds the limits, test the
contamination, preferably in a laminar-fiow cabinet. preparation by the microscopic partic\e count test.
Very carefully wash the glassware and filtration equipment Test 1.A - Solwions for infusion or solutions for injection supplied
used, except for the membrane filters, with a warm detergent in containers with a nominal content of more than 100 mL
solution and rinse with abundant amounts of water to The preparation complies with the test if the average number
remove all traces of detergent. Immediately before use, rinse of partic\es present in the units tested does not exceed 25 per
the equipment from top to bottom, outside and then inside, millilitre equal to or greater than 10 fim and does not exceed
with particle-free water R . 3 per millilitre equal to or greater than 25 ~Lm .
Take care not to introduce air bubbles into the preparation Test l.B - Solutions for infusion or solutions for injection supplied
to be examined, especially when fractions of the preparation in containers with a nominal content of less ¡han 100 mL
are being transferred to the container in which the The preparation complies with the test if the average number
of partic\es present in the units tested does not exceed
1 This chapter has undergone pharmacopoeial hannonisation. See chapter

V-A380 Appendix XIII A 2014
GFOVeirele
00 00
¡ -ot- ~ -~ - - - - - Reference
circle
00
oo •• Cross
hairs
Linear scale
11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111 -' -
Figure 2.9.19.-1. - Circular diameter graticule
6000 per container equal 10 or greater than 10 ¡lm and do es top to bottom, outside and then inside, with particle-free
not exceed 600 per container equal 10 or greater than 25 ¡lm. water R.
In order to check that the environment is suitable for the
Method 2. Microscopic partic1e count test test, that the glassware and the membrane filter are properly
Use a suitable binocular microscope, filter assembly for c1eaned and that the water 10 be used is partic1e-free, the
retaining particulate contamination and membrane filter for following test is carried out: determine the particulate
examination. 'contamination of a 50 mL volume of particle-free water R
The microscope is equipped with an ocular micro meter according to the method described below. If more than
calibrated with an objective micrometer, a mechanical stage 20 partic1es 10 ¡lm or larger in size or if more than 5 partic1es
capable of holding and traversing the entire filtration area of 25 ¡lm or larger in size are present within the filtration area,
the membrane filter, 2 suitable illumina10rs 10 provide the precautions taken for the test are not sufficient.
episcopic illuminatiort in addition to oblique illumination, The preparatory steps must be repeated until the
and is adjusted 10 100 ± 10 magnifications. environment, glassware, membrane filter and water are
The ocular micrometer is a circular diameter graticule (see suitable for the test.
Figure 2.9.19.-1.) and consists of a large circ1e divided by METHOD
crosshairs into quadrants, transparent and black reference Mix the contents of the samples by slowly inverting the
circ1es 10 ¡lm and 25 ¡lm in diameter at 100 magnifications, . container 20 times successively. If necessary, cautiously
and a linear scale graduated in 10 ¡lm increments. It is remove the sealing c1osure. Clean the outer surfaces of the
calibrated using a stage micro meter that is certified by either container opening using a jet of particle-free water R and
a domestic or international standard institution. A relative remove the c1osure, avoiding any contamination of the
error of the linear scale of the graticule within ± 2 per cent contents.
is acceptable. The large circ1e is designated the graticule field For large-volume parenterals, single units are tested.
ofview (GFOV). For small-volume parenterals less than 25 mL in volume, the
2 illuminators are required. One is an episcopic brightfield contents of lOor more units are combined in a c1eaned
illumina10r internal to the microscope, the other is an container; where justified and authorised, the test solution
external, focusable auxiliary illuminator adjustable to give may be prepared by mixing the contents of a suitable number
reftected oblique illumination at an angle of 10-20°. of vials and diluting 10 25 mL with particle-free water R or
The filter assembly for retaining particulate contamination with an appropriate solvent without contamination of
consists of a filter holder made of glass or other suitable partic1es when particle-free water R is not suitable. Small-
material, and is equipped with a vacuum source and a volume parenterals having a volume of 25 mL or more may
suitable membrane filter. be tested individually.
The membrane filter is of suitable size, black or dark grey in Powders for parenteral administration are constituted with
colour, non-gridded or gridded, and 1.0 ¡lm or finer in particle-free water R or with an appropriate solvent without
nominal pore size. contamination of partic1es when particle-free water R is not
suitable.
GENERAL PRECAUTIONS
The test is carried out under conditions lirniting particulate The number of test specimens must be adequate 10 provide a
contamination, preferably in a laminar-ftow cabinet. statistically sound assessment. For large-volume parenterals
or for small-volume parenterals having a volume of 25 mL or
Very carefully wash the glassware and filter assembly used,
more, fewer than 10 units may be tested, based on an
except for the membrane filter, with a warm detergent
appropriate sampling plan.
solution and rinse with abundant amounts of water to
remove all traces of detergent. Immediately before use, rinse Wet the inside of the filter holder fitted with the membrane
both sides of the membrane filter and the equipment from filter with several millilitres of particle-free water R. Transfer to
2014 Appendix XIII B V-A381
the filtration funnel the total volume of a solution pool or of

a single unit, and apply vacuum. If needed, add stepwise a
B. Particulate Contamination: Visible
portion of the solution until the entire volume is filtered. Particles
After the last addition of solution, begin rinsing the inner
(Ph. Eur. mechod 2.9.20)
walls of the filter holder by using a jet of particle-free water R.
Particulate contamination of injections and infusions consists
Maintain the vacuum until the surface of the membrane filter
of extraneous, mobile undissolved particles, other than gas
is free from liquid. Place the filter in a Petri dish and allow
bubbles, unintentionally present in the solutions.
the filter to air-dry with the cover slightly ajar. After the filter
has been dried, place the Petri dish on the stage of the The test is intended to provide a simple procedure for the
microscope, scan the entire membrane filter under the visual assessment of the quality of parenteral solutions as
reflected light from the illuminating device, and count the regards visible particles. Other validated methods may be
number of particles that are equal to or greater than 10 Jlm used.
and the number of particles that are equal to or greater than Apparatus
25 Jlm. Alternatively, partial fiiter count and determination of
the total filter count by calculation is allowed. Calculate the The apparatus (see Figure 2.9.20.-1) consists of a viewing
station comprising:
mean number of particles for the preparation to be
examined. - a matt black panel of appropriate size held in a vertical
The particle sizing process with the use of the circular position,
diameter graticule is carried out by transforming mentally the - a non-glare white panel of appropriate size held in a
image of each particle into a circle and then comparing it to vertical position next to the black panel,
the 10 Jlm and 25 Jlm graticule reference circles. Thereby the - an adjustable lampholder fitted with a suitable, shaded,
particles are not moved from their initial locations within the white-light source and with a suitable light diffuser (a
graticule field of view and are nor superimposed on the viewing illuminator containing two 13 W fluorescent
reference circles for comparison. The inner diameter of the tubes, each 525 mm in length, is suitable). The intensity
transparent graticule reference circles is used to size white of illumination at the viewing point is maintained between
and transparent particles, while dark particles are sized by 2000 lux and 3750 lux, although higher values are
using the outer diameter of the black opaque graticule preferable for coloured glass and plastic containers.
reference circles.
Adjustable lampholder
In performing the microscopic particle count test do not
attempt to size or enumerate amorphous, semi-liquid, or /'
otherwise morphologically indistinct materials that have the
appearance of a stain or discoloration on the membrane
fiiter. These materials show little or no surface relief and
present a gelatinous or film-like appearance. In such cases
Malt
the interpretation of enumeration may be aided by testing a black
sample of the solution by the light obscuration particle count panel
test.
EVALUATION
volume of more than 100 mL, apply the criteria of test 2.A.
Figure 2.9.20.-1. - Apparatus for visible particles
volume ofless than 100 mL, apply the criteria oftest 2.B.
For preparations supplied in containers with a nominal Method
volume of 100 mL, apply the criteria of test 2.B. Remove any adherent labels from the container and wash
Test 2.A - Solutions for infusion or solutions for injection supplied and dry the outside. Gently swirl or invert the container,
in containers with a nominal content of more than 100 m L ensuring that air bubbles are not introduced, and observe for
about 5 s in front of the white panel. Repeat the procedure
The preparation complies with the test if the average number
in front of the black panel. Record the presence of any
of particles present in the units tested does not exceed 12 per
particles.
millilitre equal to or greater than 1O ~lm and does not exceed
2 per millilitre equal to or greater than 25 Jlm .
Test 2.B - Solutions for infLlSion or solutions for injection supplied
in containers with a nominal content of less than 100 mL
The preparation complies with the test if the average number
of particles present in the units tested does not exceed
3000 per container equal to or greater than 10 Jlm and do es
not exceed 300 per container equal to or greater than 25 Jlm.
V-A382 Appendix XIV 2014
In order to assess the validity of the assay, use not fewer than
Appendix XIV 3 doses of the reference substance and 3 doses of the
antibiotic to be examined having the same presumed activity
as the doses of the reference substance. It is preferable to use
Biological Assays and Tests a series of dos es in geometric progression. In routine assays
General guidance conceming biological assays and tests is provided when the linearity of the system has been demonstrated over
in Supplementary Chapter 1 H. an adequate number of experiments using a three-point
assay, a two-point assay may be sufficient, subject to
agreement by the competent authority. However, in all cases
of dispute, a three-point assay as described aboye must be
A. Microbiological Assay of Antibiotics applied.
Arrange the solutions on each Petri dish or on each
rectangular dish according to a statistically suitable design,
The potency of an antibiotic is estimated by comparing the
except for small Petri dishes that cannot accommodate more
inhibition of growth of sensitive micro-organisms produced
than 6 solutions, arrange the solutions of the antibiotic to be
by known concentrations of the antibiotic to be examined
examined and the solutions of the reference substance in an
and a reference substance.
alternate manner to avoid interaction of the more
The reference substances used in the assays are substances concentrated solutions.
whose activity has been precisely determined with reference
Incubate at a suitable temperature for about 18 h . A period
to the corresponding international standard or international
of diffusion prior to incubation, usually 1-4 h, at room
reference preparation.
temperature or at about 4 oC, as appropriate, may be used to
The assay must be designed in a way that will permit minimise the effects of the variation in time between the
examination of the validity of the mathematical model on application of the solutions and to improve the regression
which the potency equation is based. If a parallel-line model slope.
is chosen, the 2 log dose-response (or transformed response)
Measure the diameters with a precision of at least 0.1 mm or
lines of the preparation to be examined and the reference
the areas of the circular inhibition zones with a
preparation must be parallel; they must be linear over the
corresponding precision and calculate the potency using
range of doses used in the calculation. These conditions must
llPpropriate statistical methods.
be verified by validity tests for a given probability, usually
P = 0.05 . Other mathematical models, such as the slope Use in each assay the number of replications per dose
ratio model, may be used provided that proof of validity is sufficient to ensure the required precision. The assay may be
demonstrated. repeated and the results combined statistically to obtain the
required precision and to ascertain whether the potency of
Unless otherwise stated in the monograph, the confidence
the antibiotic to be examined is not less than the minimum
limits (P = 0.95) of the assay for potency are not less than
required.
95 per cent and not more than 105 per cent of the estimated
potency. B. TURBIDlMETRIC METHOD
Carry out the assay by method A or method B.
Inoculate a suitable medium with a suspension of the chosen
A. DIFFUSION METHOD micro-organism having a sensitivity to the antibiotic to be
examined such that a sufficiently large inhibition of microbial
LiquefY a medium suitable for the conditions of the assay growth occurs in the conditions of the test. Use a known
and inoculate it at a suitable temperature, for example 48 oC quantity of the suspension chosen so as to obtain a readily
to 50 oC for vegetative forms, with a known quantity of a measurable opacity after an incubation period of about 4 h.
suspension of micro-organisms sensitive to the antibiotic to
Use the inoculated medium immediately after its preparation .
be examined, such that clearly defined zones of inhibition of
suitable diameter are produced with the concentrations of the Using the solvent and the buffer solution indicated in
antibiotic used for the assay. Irnmediately pour into Petri Table 2.7.2.-2 prepare solutions of the reference substance
dishes or large rectangular dishes a quantity of the inoculated and of the antibiotic to be examined having known
medium to form a uniform layer 2-5 mm thick. Alternatively, concentrations presumed to be of equal activity.
the medium may consist of 2 layers, only the upper layer In order that the validity of the assay may be assessed, use
being inoculated. not fewer than 3 dos es of the reference substance and 3
Store the dishes so that no appreciable growth or death of doses of the antibiotic to be examined having the same
the micro-organisms occurs before the dishes are used and so presumed activity as the doses of the reference substance.
that the surface of the medium is dry at the time of use. It is preferable to use a series of doses in geometric
progression. In order to obtain the required linearity, it may
Using the solvent and the buffer solution indicated in
be necessaty to select from a large number 3 consecutive
Table 2.7.2.-1, prepare solutions ofthe reference substance
doses, using corresponding doses for the reference substance
and of the antibiotic to be examined having known
and the antibiotic to be examined.
concentrations and presumed to be of equal activity. Apply
the solutions to the surface of the medium, for example, in Distribute an equal volume of each of the solutions into
sterile cylinders of porcelain, stainless steel or other suitable identical test-tubes and add to each tube an equal volume of
material, or in cavities prepared in the agar. The same inoculated medium (for example, 1 mL of the solution and
volume of solution must be added to each cylinder or cavity. 9 mL of the medium). For the assay of tyrothricin add
Alternatively, use sterile absorbent paper discs of suitable 0.1 mL ofthe solution to 9.9 mL ofinoculated medium.
quality; impregnate the discs with the solutions of the Prepare at the same time 2 control tubes without antibiotic,
reference substance or the solutions of the antibiotic to be both containing the inoculated medium and to one of which
examined and place on the surface of the agar. is added immediately 0.5 mL ofjormaldehyde R. These tubes
2014 Appendix XIV A V-A383
Table 2.7.2.-1. - Diffusion assay

Solvent to be used in
Medium and final pH Incubation
Antibiotic Reference substance preparing the stock Buffer solution (pH) Micro-organism
(± 0. 1 pH unit) temperature
solution
Saccharomyces
Amphotericin B cerevisiae
Arnphotericin B for microbioloyical Dimethyl sulfoxide R pH 10.5 (0.2 M) F· pH 6.1 35-37 oC
ATCC 9763
assay CRS
IP 1432·83
Micrococcus luteus
0.01 M hydrochloric NCTC 7743
Bacitracin zinc Bacitracin zinc CRS pH 7.0 (0.05 M) A·pH 7.0 35·39 oc
acid CIP 53.160
ATCC 10240
Mycobacterium
Bleomycin smegmatis
Bleornycin sulfate WaterR pH 6.8 (0.1 M) G· pH 7.0 35.37 oC
sulfate CRS
ATCC 607
Bordetella B· pH 7.3 35.39 oC
bronchiseptica
NCTC 8344
ClP 53.157
Colistimethate Colistimethate
WaterR pH 6.0 (0.05 M) ATCC 4617
sodiurn sodium CRS
Escherichia coli B· pH 7.3 35·39 oC
NCIB 8879
ClP 54.127
ATCC 10536
Bordetella B· pH 7.3 35.39 oC
.bronchiseptica
NCTC 8344
CIP 53.157
Colistin sulfate for
Colistin sulfate microbiological Water R pH 6.0 (0.05 M) ATCC 4617
assay CRS Escherichia coli B· pH 7.3 35·39 oC
NCIB 8879
ClP 54.127
ATCC 10536
Bacillus subtilis E· pH 7.9 30·37 oC
NCTC 10400
ClP 52.62
Framycetin ATCC 6633
Framycetin sulfate WaterR pH 8.0 (0.05 M)
sulfate CRS
Bacillus pumilus E· pH 7.9 30·37 oC
NCTC 8241
ClP 76.18
Bacillus pumilus A· pH 7.9 35.39 oC
NCTC 8241
ClP 76.18
Gentamicin Staphylococcus A·pH 7.9 35-39 oC
Gentarnicin sulfate Water R pH 8.0 (0.05 M)
sulfate CRS epidermidis
NCIB 8853
ClP 68.21
ATCe 12228
Bacillus subtilis
Methanol R (see the elP 52.62
Josarnycin Josamycin CRS pH 5.6 A- pH 6.6 35-37°e
rnonograph) ATCC 6633
NCTe 10400
Bacillus subtilis
Josamycin Methanol R (see the ClP 52.62
Josarnycin propionate pH 5.6 A· pH 6.6 35-37 °e
propionate CRS rnonograph) ATee 6633
NCTC 10400
Bacillus subtilis A - pH 7.9 30-37 oC
Kanarnycin NCTe 10400
rnonosu lfate elP 52.62
ATCC 6633
Kanamycin
Water R pH 8.0 (0.05 M) Staphylococcus A-pH 7.9 35·39°e
monosulfate CRS
aureus
Kanarnycin acid NCTe 7447
sulfate
CIP 53.156
ATee 6538 P
V-A384 Appendix XIV A 2014
Solvent to be used in Medium and final pH Incubation

Antibiotic Reference substance preparing the stock Buffer solution (pH) Micro-organism (± 0.1 pH unít) temperature
solution
Bacillus pumilus E - pH 7.9 30-37 oC
NCTC 8241
CIP 76.18
Neomycin sulfate
Neomycin sulfate for microbiological Water R pH 8.0 (0 .05 M) Bacillus subtilis E - pH 7.9 30-37 oC
assay CRS
NCTC 10400
CIP 52.62
ATCC 6633
Slaphylococcus
Netilmicin aureus
Netilmicin sulfate Wa terR pH 8.0 ± 0.1 A - pH 7.9 32-35 oC
sulfate CRS ATCC 6538 P
CIP 53.156
Candida tropicalis F- pH 6.0 30-37 oC
CIP 1433-83
NCYC 1393
pH 6.0 (0.05 M) con-
Dimethylforma- taining 5 per cent V/V Saccharomyces F- pH 6.0 30-32 oc
Nystatin Nystatin CRS
mideR of dim ethylforma- cerevisiae
mideR
NCYC 87
CIP 1432-83
ATCC 9763
Micrococcus luteus
Rifamycin NCTC 8340
Rifamycin sodium Methanol R pH 7.0 (0.05 M) A- pH 6.6 35-39 oC
sodium CRS CIP 53.45
ATCC 9341
Bacillus subtilis
NCTC 10400
Spiramycin Spiramycin CRS Methanol R pH 8.0 (0.05 M) A- pH 7.9 30-32 oC
CIP 52.62
ATCC 6633
Bacillus subtilis A-pH7. 9 30-37 oC
NCTC 8236
CIP 1.83
Streptomycin
Streptomycin sulfate sulfate CRS Water R pH 8.0 (0.05 M) Bacillus subtilis A-pH 7.9 30-37 oC
-
NCTC 10400
CIP 52.62
ATCC 6633
Bacillus subtilis
NCTC 10400
Teicoplanin Teicoplanin CRS pH 6.0 (0.05 M) pH 6.0 (0.05 M) H - pH 7.8·8.0 35-37 oC
CIP 52.62
ATCC 6633
Tylosin for veterinary A mixture of
2.5 per cent V/V Micrococcus luteus
use 40 volumes of
solution of NCTC 8340
methanol R and
Tylosin CRS methanol R in 0.1 M A - pH 8.0 32-35 oC
phosphate buffer
60 volumes of 0.1 M CIP 53.45
Tylosin tartrate for phosphale buffer
veterinary use solution pH 7.0 R ATCC 9341
solu/ion pH 8.0 R
Bacillus subtilis
Vancomycin Vancomycin NCTC 10400
hydrochloride hydrochloride CRS
WalerR pH 8.0 A-pH 8.0 37-39 oC
CIP 52.62
ATCC 6633
are used ro set the optical apparatus used to measure the which allows the opacity of each tube ro be measured after
growth. exactly the same period of incubation.
Place an the tubes, randomly distributed or in a L atin square Calculate the potency using appropriate statistical methods .
or randomised block arrangement, in a water-bath or other Linearity of the dose-response relationship, transformed or
suitable apparatus fitted with a means of bringing all the untransforrned, is often obtained only over a very limited
tubes rapidly ro the appropriate incubation temperature and range. Ir is this range which must be used in calculating the
maintain them at that temperature for 3-4 h, taking activity and it must include at least 3 consecutive doses in
precautions to ensure uniforrnity of temperature and identical order ro permit linearity to be verified. In routine assays
incubation time. when the linearity of the system has been demonstrated over
After incubation, stop the growth of the micro-organisms by an adequate number of experiments using a three-poiiu
adding 0.5 mL ofjonnaldehyde R to each tube or by heat assay, a two-point assay may be sufficient, subject to
treatment and measure the opacity ro 3 significant figures agreement by the competent authority. H owever, in all cases
using suitable optical apparatus. Altematively use a method of dispute, a three-point assay must be applied.
Table 2.7.2.-2. - Turbidimetric assay

Solvent to be used in
Medium and final pH
Antibiotic Reference substance preparing the stock Buffer solution (pH) Micro·organism Incubation temperature
(± 0.1 pH unit)
solution
Escherichia coa
eolistimethate Coastimethate NelB 8666
WaterR pH 7.0 e· pH 7.0 35·37°e
sodium sodium CRS elP 2.83
ATee 9637
Escherichia coa
Colistin sulfate for NelB 8666
eolistin sulfate microbiological Water R pH 7.0 e · pH 7.0 35·37°e
assay CRS elP 2.83
ATee 9637
Staphylococcus
aureus
Framycetin NCTe 7447
Framycetin sulfate
sulfate CRS WaterR pH 8.0 e· pH 7.0 35·37 °e
elP 53.156
ATee 6538 P
Staphylococcus
aureus
Gentamicin sulfate
Gentamicin WaterR pH 7.0 NCTe 7447 e· pH 7.0
sulfate CRS 35·37°e
elP 53.156
ATee 6538 P
Enterococcus hirae
elP 58.55
ATee 10541
Gramicidin CRS MethanolR pH 7.0' e· pH 7.0 35.37 °e
Staphylococcus
Gramicidin aureus
ATee 6538 P
'Addition of a detergent may be necessary to avoid adsorption on the material during the dilutions. for example 0.1 mg¡'mL
of polysorbate 80 R
Staphylococcus
aureus
Josamycin Josamycin CRS Me/hanol R (see the pH 5.6 elP 53.156 e· pH 8.0 35·37 °e
monograph)
ATee 6538 P
NCTe 7447
Staphylococcus
aureus
Josamycin propionate
Josamycin Methanol R (see the pH 5.6 elP 53.156 35.37 °e
e· pH 8.0
propionate CRS monograph)
ATee 6538 P
NeTe 7.447
Kanamycin Staphylococcus
monosulfate aureus
Kanamycin NeTe 7447
monosulfate CRS Wa/erR pH 8.0 e·pH 7.0 35.37 °e
Kanamycin acid elP 53.156
sulfate ATee 6538 P
S/aphylococcus
Neomycin sulfa/e aureus
Neomycin sulfate for microbiological Water R pH 8.0 NCTe 7447 e· pH 7.0 35·37°e
assay CRS elP 53.156
ATee 6538 P
Escherichia coli
Rifamycin NelB 8879
Rifamycin sodium Me/hanol R pH 7.0 e· pH 7.0 35·37°e
sodium CRS elP 54.127
ATee 10536
Staphylococcus
aureus
Spiramycin Spiramycin CRS Me/hanol R pH 7.0 NCTe 7447 e· pH 7.0 35·37°e
e lP 53.156
ATee 6538 P
Klebsiella
pneumoniae
Streptomycin NeTe 7427
Streptomycin sulfate
sulfate CRS WaterR pH 8.0 e· pH 7.0 35·37 °e
elP 53.153
ATee 10031
V-A386 Appendix XIV A 2014
Solvent to be used in Medium and final pH

Antibiotic Reference substance preparing the stock Buffer solution (pH) Micro-organism Incubation temperature
(± 0.1 pH unit)
solution
Tylosin for veterinary 2.5 per cent VjV Staphylococcus
use solution of aureus
Tylosin CRS methanol R in 0.1 M pH 7.0 NCTC 6571 C- pH 7.0 37 oC
Tylosin tartrate for phosphate buffer ATCC 9144
veterinary use solution pH 7. O R CIP 53.154
Enterococcus hirae
Tyrothricin Cramicidin CRS Alcohol R Alcohol R C - pH 7.0 37 oC
ATCC 10541
Staphylococcus
Vancornycin Vancomycin aureus
Water R pH 8.0 C - pH 7.0 37-39 oC
hydrochloride hydrochloride CRS CIP 53.156
ATCC 6538 P
Use in each assay me number of replications per dose Saccharomyces cerevisiae; Candida tropicalis Grow
sufficient 10 ensure me required precision. The assay may be the test organism on medium F at 30-37 oC for 24 h. Wash
repeated and me results combined statistically 10 obtain me off me growth wim a sterile 9 gIL solution of sodium
required precision and 10 ascertain whemer the potency of chloride R. Dilute to a suitable opacity wim the same
the antibiotic to be examined is not les s man the minirnum solution.
required. Buffer solutions Buffer solutions having a pH between
The following section is published for information. 5.8 and 8.0 are prepared by mixing 50.0 mL of 0.2 M
Recommended micro-organisms potassium dihydrogen phosphate R with me quantity of 0.2 M
sodium hydroxide indicated in Table 2.7.2.-3. Dilute with
The following text details the recommended micro-organisms freshly prepared distilled water R to produce 200 .0 mL.
and the conditions of use. Other micro-organisms may be
used provided mat they are shown to be sensitive to me Table 2.7.2.-3.
antibiotic to be examined and are used in appropriate media
and appropriate conditions of temperature and pH. pH 0.2 M Sodium hydroxide (mL)
The concentrations of the solutions used should be chosen so 5.8 3.72
as 10 ensure mat a linear relationship exists between me
6.0 5.70
logarithm of me dose and me response in the CGnditions of
the test. 6.2 8.60
Preparation of inocula Bacillus cereus varo mycoides; 6.4 12.60
Bacillus subtilis; Bacillus pumilus. Spore suspensions of the
6.6 17.80
organisms 10 be used as inocula are prepared as follows.
6.8 23.65
Grow the organism at 35-37 oC for 7 days on the surface of
a suitable medium to which has been added 0.001 gIL of 7.0 29.63
manganese sulfate R. Using sterile water R, wash off the 7.2 35.00
growth, which consists mainly of spores. Heat me suspension
at 70 oC for 30 min and dilute to give an appropriate 7.4 39.50
concentration of spores, usually 10 x 106 10 100 x 10 6 per 7.6 42.80
millilitre. The spore suspensions may be stored for long
7.8 45.20
periods at a temperature not exceeding 4 oC.
8.0 46.80
Altematively, spore suspensions may be prepared by .
cultivating the organisms in medium e at 26 oc for 4-6 days,
then adding, aseptically, sufficient manganese sulfate R to give These buffer solutions are used for aH microbiological assays
a concentration of 0.001 gIL and incubating for a further shown in Table 2.7.2.-1 with the exception ofbleomycin
48 h. Examine the suspension microscopically to ensure mat sulfate and amphotericin B.
adequate spore formation has taken place (about 80 per cent) For bleomycin sulfate, prepare the buffer solution pH 6.8 as
and centrifuge. Re-suspend the sediment in sterile water R to follows: dissolve 6.4 g of potassium dihydrogen phosphate R and
give a concentration of 10 x 10 6 to 100 x 10 6 spores per 18.9 g of disodium hydrogen phosphate R in water R and dilute
millilitre, and then heat to 70 oC for 30 mino Store me to 1000 mL with water R.
suspension at a temperature not exceeding 4 oc.
For amphotericin B, prepare the 0.2 M phosphate buffer
Bordetella bronchiseptica Grow me test organism on solution pH 10.5 as foHows: dissolve 35 g of dipotassium
medium B at 35-37 oC for 16-18 h. Wash offthe bacterial hydrogen phosphate R in 900 mL of water R, add 20 mL of
growth with sterile water R and dilute to a suitable opacity. 1 M sodium hydroxide and dilute to 1000.0 mL with water R.
Staphylococcus aureus; Klebsiella pneumoniae; Culture media The following media or equivalent media
Escherichia coli; Micrococcus luteus; Staphylococcus may be used.
epidermidis Prepare as described aboye for B.
bronchiseptica but using medium A and adjusting me opacity
to one which has been shown to produce a satisfactory dose-
response relationship in me turbidimetric assay, or to
produce c1early defined zones of inhibition of convenient
diameter in me diffusion assay, as appropriate.
Method A. Diffusion Method

Table 2.7.2.-1 - Diffusion Assay; Additional Section for Monographs of the British Pharmacopoeia
Antibiotic Reference Solvent to Buffer Micro-organism Medium and Incubation

substance be used in solution final pH (± 0.1 temperature
preparing (pH) pH unit)
the stock
Solution
Erythrornycin Bacillus pumilus A; 7.9 30 - 37°

Estolate NCTC 8241
CIP 76.18
Erythrornycin Ethyl Erythromycin Methanol pI-! 8.0
Succinate EPCRS (0.05M) Bacillus subtilis A; 7.9 30 - 37°
NCTC 10400
Erythrornycin CIP 52.62
Stearate ATCC 6633
Lyrnecycline Lyrnecycline pI-! 5.8 Bacillus pumilus A; 6.6 37 - 39°

2 nd Int. Ref., NCTC 8241
1971 CIP 76.18
Polyrnyxin B Polymyxin B Water pI-! 6.0 Bordetella B;7.3 35 - 39°

Sulphate Sulphate EPCRS (0.05M) bronchiseptica
NCTC 8344
CIP53.157
ATCC4617
Method B. Turbidimetric Method

Table 2.7.2.-2 - Turbidimetric Assay; Additional Sectionfor Monographs ofthe British Pharmacopoeia
Antibiotic Reference Solvent to Buffer Micro-organism Medium and Incubation

substance be used in solution final pH (± 0.1 temperature
preparing (pH) pH unit)
the stock
Solution
Aprarnycin Apramycin BPCRS * Sa/monella 1; 8.0 37°

cholerasuis
Erythrornycin Klebsiella D; 7.0 35 - 37°

Estolate pneumoniae
NCTC 7427
Erythrornycin Ethyl Erythromycin Methano/" pI-! 8.0 CIP 53.153
Succinate EPCRS ATCC 10031
C; 7.0 35 - 37°
Erythrornycin Staphy/ococcus
Stearate aureus
NCTC 7447
CIP 53 .156
ATCC 6538 P
* Solution prepared by dissolving 16.73 g of dipotassium hydrogen orthophosphate and 0.523 g of potassium dihydrogen
orthophosphate in about 750 mI of water, if neeessary adjusting to pH 8.0 with O.lM sodium hydroxide or O.lM orthophosphoric acid,
and diluting to 1000 mI with water.
MediumA MediumB
Peptone 6g Panereatic digest of easein 17 g
Panereatic digest of easein 4g Papaic digest of soya bean 3g
Beef extraet l.5 g Sodium ehloride 5g
Yeast extraet 3g Dipotassium hydrogen phosphate 2.5 g
Glueose monohydrate 1g Glueose monohydrate 2.5 g
Agar 15 g Agar 15 g
Water to 1000 mL Polysorbate 80 10 g
Water to 1000 mL
V-A388 Appendix XIV B 2014
The polysorbate 80 is added 10 the hot solution of the other basis for ealculating the quantity of an antibiotie 10 be
ingredients after boiling, and immediately before adjusting to ineorporated in a preparation. In sueh eireumstanees, assays
volurne. of greater preeision may be desirable with, for instanee,
fiduciallimits oferror ofthe order of98 10 102%. With this
Medium C
degree of preeision, the lower fidueiallimit líes close 10 the
Peptone 6 g
estimated poteney. By using this limit, instead of the
Beef extraet 1.5 g
estimated poteney, to assign a poteney to the antibiotie either
Yeast extraet 3g
for labelling or for ealculating the quantity 10 be included in
Sodiurn ehloride 3.5 g
a preparation, there is less líkelihood of the final preparation
Glueose monohydrate 1g
subsequently failing 10 eomply with the offieial requirements
Dipotassium hydrogen phosphate 3.68 g
for poteney.
Potassium dihydrogen phosphate 1.32 g
Water to 1000 mL Culture Media The following media or equivalent media
may be used.
MediumD
Medium 1
Heart extraet 1.5 g
D-Glueose 10 g
Yeast extraet 1.5g
Tryp10ne 6 g
Pep1One-easein 5g
Yeast extraet* 2g
Glueose monohydrate 1g
Water 10 produce 1000 mL
Sodiurn ehloride 3.5 g
Dipotassium hydrogen phosphate 3.68 g Adjust to pH 8.0 with 1M sodium hydroxide or
Potassiurn dihydrogen phosphate 1.32 g O.IM onhophosphoric acid.
Potassium nitrate 2g (*Ardamine yeast extraet supplíed by Champlaín Industries
Water to 1000 mL Ine., Clifton, NJ 07012, USA is suitable.)
Medium E
Peptone 5g
Meat extraet 3g
Disodium hydrogen phosphate,12H 2 0 26.9 g B. Immunochemical Methods
Agar 10 g (Ph. Eur. mechod 2.7.1)
Water to 1000 mL Immunoehemieal methods are based on the seleetive,
The disodium hydrogen phosphate is added as a sterile reversible and non-eovalent bínding of antigens by
solution after sterilisation of the medium. antibodies. These methods are employed to deteet or
quantifY either antigens or antibodies. The formation of an
MediumF
antigen-antibody eomplex may be deteeted, and the amount
Peptone 9.4 g
of eomplex formed may be measured by a variety of
Yeast extraet 4.7 g
teehniques. The provisions of this general method apply 10
Beef extraet 2.4 g
irnmunoehemieal methods using labelled or unlabelled
Sodium ehloride 10.0 g
reagents, as appropriate.
Glueose monohydrate 10.0 g
Agar 23.5 g The results of immunoehemical methods depend on the
Water to 1000 mL experimental eonditions and the nature and quality of the
reagents used. It is essential 10 standardise the eomponents of
Medium G an irnmunoassay and to use, wherever available, intemational
Glyeerol 10 g referenee preparations for immunoassays.
Pep10ne 10 g
The reagents neeessary for many immunoehemieal methods
Meat extraet 10 g
are available as eommereial assay kits, that is, a set including
Sodiurn ehloride 3g
reagents (partieularly the antigen or the antibody) and
Agar 15 g
materials intended for the in viero estimation of a speeified
Water 10 1000 mL
substanee as well as instruetions for their proper use.
pH 7.0 ± 0.1 after sterilisation. The kits are used in aeeordanee with the manufaeturers'
Medium H instruetions; it is important to aseertaín that the kits are
Peptone 5.0 g suitable for the analysis of the substanee to be examined,
Agar 15.0 g with particular referenee 10 seleetivity and sensitivity.
Beef extraet powder 3.0 g Guidanee eoneemíng irnmunoassay kits is provided by the
Water to 1000 mL World Health Organisation, Teehnical Report Series 658
(1981).
pH 7.8 - 8.0 adjusted with 0.1 M sodium hydroxide.
Methods in which a labelled antigen or a labelled
Monographs o/ the British Pharmacopoeia antibody is used
The following additional information and guidanee apply to
Methods using labelled substanees may employ suitable
monographs of the British Pharmaeopoeia.
labels sueh as enzymes, fluorophores, luminophores and
The required mínimum preeision for an aeeeptable assay of radioisotopes . Where the label is a radioiso1Ope, the method
any particular antibiotie or preparation is defined in the is deseribed as a " radio-immunoassay" .
appropriate monograph in the paragraph on the Assay. This The reeommendations for the measurement of radioaetivity
degree of preeision is the mínimum aeeeptable for given in the monograph on Radiopharmaceutical Preparations
determining that the final produet eomplíes with the offieial (0125) are applieable to irnmunoassays involving
requirements. It may be inadequate for a deeision about the radioiso1Opes . AH work with radio active materials must be
poteney that should be stated on the labe! or used as the earried out in eonformity with national legislation and
2014 Appendix XIV B V-A389
internationally accepted codes of practice for protection the second direction is carried out perpendicular to the
against radiation hazards. previous electrophoretic run in a gel containing a
comparatively low concentration of antibodies corresponding
Methods in which an unlabelled antigen or antibody ro the antigens. For a given antibody concentration and gel
is used thickness, the relationship between the area of the respective
lmmunopreeipitation methods precipitation peaks and the amount of the corresponding
Immunoprecipitation methods inelude ftocculation and antigen is linear.
precipitation reactions. When a solution of an antigen is Electroi111munoassay, often referred to as rocket im111uno-
mixed with its corresponding antibody under suitable electrophoresis is a rapid quantitative method for determining
conditions, the reactants form ftocculating or precipitating antigens with a charge differing from that of the antibodies or
aggregates. The ratio of the reactants which gives the shortest vice versa. The electrophoresis of the antigen ro be
ftocculation time or the most marked precipitation is called determined is carried out in a gel containing a comparatively
the optimal ratio, and is usually produced by equivalent lower concentration of the corresponding antibody. The test
amounts of antigen and antibody. Immunoprecipitation can material and dilutions of a standard antigen used for
be assessed visually or by light-scattering techniques calibration are introduced into different wells in the gel.
(nephelometric or turbidimetric assay) . An increase in During electrophoresis, migrating peak-shaped precipitation
sensitivity can be obtained by using antigen- or antibody- zones originating from the wells are developed. The front of
coated partieles (e.g. latex) as reactants . the precipitate becomes stationary when the antigen is no
In ftocculation methods, stepwise dilutions of one of the longer in excess. For a given antibody concentration, the
reactants is usually used whereas, in immunodiffusion (ID) relationship between the distan ce travelled by the precipitate
methods, the dilution is obtained by diffusion in a gel and the amount of antigen applied is linear.
medium : concentration gradients of one or both of the Counter-im111unoelectrophoresis is a rapid quantitative method
reactants are obtained, thus creating zones in the gel medium allowing concentration gradients of external antigen and
where the ratio of the reactants favours precipitation. While external antibody to be established in an electric field
ftocculation methods are performed in tubes, depending on the different charges. Dilutions of a standard
immunodiffusion methods may be performed using different for calibration and dilutions of the test material are
supports such as tubes, plates, slides, cells or chambers. introduced into a row of wells in a gel and a fixed amount of
Where the immunoprecipitating system consists ofone the corresponding reactant is introduced into an opposite row
antigen combining with its corresponding antibody, the of wells. The titre of the test material may be determined as
system is referred to as simple; when it involves related but the highest dilution showing a precipitation lineo
not serologically identical reactants, the system is complex and A number of modifications of crossed immunoelectrophoresis
where several serologically unrelated reactants are involved, and electroimmunoassay methods existo
the system is multiple. Other techniques combine separation of antigens by
In simple diffusion methods, a concentration gradient is molecular size and serological properties.
established for only one of the reactants diffusing from an Visualisation and eharaeterisation 01
externa] source into the gel medium containing the immunopreeipitation lines
corresponding reactant at a comparatively low concentration.
These may be performed by selective or non-selective stains,
Single radial immunodiffusion (SRID) is a simple quantitative by ftuorescence, by enzyme or isotope labelling or other
immunodiffusion technique. When the equilibrium between relevant techniques. Selective staining methods are usually
the external and the internal reactant has been established, performed for characterisation of non-protein substances in
the circular precipitation are a, originating from the site of the the precipita tes.
external reactant, is directly proportional to the amount of
In translucent gels such as agar or agarose, the precipitation
the antigen applied and inversely proportional to the
line becomes elearly visible in the gel, provided that the
concentration of the antibody in the gel.
concentration of each of the reactants is appropriate.
In double diffusion methods, concentration gradients are
established for both reactants. Both antigen and antibody Validation ofthe method
diffuse from separate sites into an initially immunologically Validation eriteria
neutral gel.
A quantitative immunochemical method is not valid unless:
Comparative double diffusion methods are used for qualitatively
1) The antibody or antigen does not significantly
comparing various antigens versus a suitable antibody or vice
discriminate between the test and standard. For a
versa. The comparison is based on the presence or absence
of interaction between the precipitation patterns. Reactions of labelled reactant, the corresponding reactant do es not
identity, non-identity or partial identity of antigens/antibodies significantly discriminate between the labelled and
can be distinguished. unlabelled compound,
2) The method is not affected by the assay matrix, that is,
lmmunoeleetrophoretie methods any component of the test sample or its excipients,
Immunoelectrophoresis (lE) is a qualitative technique which can vary between samples. These may inelude
combining 2 methods: gel electrophoresis followed by high concentrations of other proteins, salts, preservatives
immunodiffusion. or contaminating proteolytic activity,
Crossed immunoelectrophoresis is a modification of the lE 3) The limit of quantitation is below the acceptance criteria
method. It is suitable both for qualitative and quantitative stated in the individual monograph,
analysis. The first part of the procedure is an ordinary gel 4) The precision of the assay is such that the variance of
electrophoresis, after which a longitudinal gel strip, the results meets the requirements stated in the
containing the separated fractions ro be determined, is cut individual monographs,
out and transferred to another plateo The electrophoresis in
V-A390 Appendix XIV e 2014
5) The order in which the assay is performed do es not give The test is carried out in a manner that avoids endotoxin
rise to systematic errors. contamination.
Validation methods 1. Apparatus
In order to verify these criteria, the validation design ineludes
Depyrogenate all glassware and other heat-stable apparatus in
the following elements:
a hot-air oven using a validated process. A commonly used
1) The assay is performed at least in triplicate, minimum time and temperature is 30 minutes at 250 oc.
2) The assayineludes at least 3 different dilutions of the If employing plastic apparatus, such as microtitre plates and
standard preparation and 3 dilutions of sample pipette tips for automatic pipetters, use apparatus shown to
preparations of presumed activity similar to the standard be free of detectable endotoxin and which do es not interfere
preparation, in the test.
3) The assay layout is randomised, NOTE: in this chapter, the term 'tube' includes al! types of
4) If the test sample is presented in serum or formulated receptacles, for example microtitre plate wells.
with other components, the standard is likewise
prepared, 2. Reagents, test solutions
5) The test ineludes the measurement of non-specific (1) Amoebocyte lysate Amoebocyte lysate is a lyophilised
binding of the labelled reactant, product obtained from amoebocyte lysate from the horseshoe
crab (Limulus polyphemus or Tachypleus tridentatus). This
6) For displacement immunoassay:
reagent refers only to a product manufactured in accordance
(a) maximum binding (zero displacement) is with the regulations of the competent authority.
determined,
NOTE: amoebocyte lysate reacts with some {J-glucans in addition
(b) dilutions cover the complete response range from to endotoxins. Amoebocyte lysate preparations which do not react
values elose to non-specific binding to maximum with glucans are available; they are prepared by removing from
binding, preferably for both standard and test amoebocyte lysate the G factor, which reacts with glucans, or by
preparations. inhibiting the G factor reacting system of amoebocyte lysate. These
Statistical calculation
preparations may be used for endotoxin testing in the presence of
glucans.
To analyse the results, response curves for test and standard
(2) Lysate solution Dissolve amoebocyte lysate in water
may be analysed by the methods described in 5.3. Statistical
for BET or in a buffer, as recommended by the lysate
Analysis of Results of Biological Assays and Tests. .
manufacturer, by gentle stirring. Store the reconstituted
Significant non-parallelism indicates that the antibody or lysate, refrigerated or frozen, as indicated by the
antigen discrimina tes between test and standard, and the manufacturero
results are not valido
(3) Water for BET (water for bacterial endotoxins test)
In displacement irnmunoassays, the values for non-specific Water for injections R or water produced by other procedures
binding and maximum displacement at high test or standard that shows no reaction with the lysate employed at the
concentration must not be significantly different. Differences detection limit of the reagent.
may indicate effects due to the matrix, either inhibition of
binding or degradation of tracer. 3. Preparation of the standard endotoxin stock
solution
The standard endotoxin stock solution is prepared from an
C. Test for Bacterial Endotoxins endotoxin reference standard that has been calibrated against
the International Standard, for example endotoxin
(LAL Test) standard BR?
(Ph. Eur. method 2.6.14) Endotoxin is expressed in Intemational Units (IU).
The test for bacterial endotoxins (BET) is used to detect or The equivalence in IU of the International Standard is stated
quantify endotoxins from gram-negative bacteria using by the World Health Organisation.
amoebocyte lysate from the horseshoe crab (Limulus NOTE: one International Unit (IU) of endolOxin is equal lO one
polyphemus or Tachypleus tridentatus). There are 3 techniques EndolOxin Unit (E. U.).
for this test: the gel-elot technique, which is based on gel
Follow the specifications in the package leaftet and on the
formation; the turbidimetric technique, based on the
labe! for preparation and storage of the standard endotoxin
development of turbidity after eleavage of an endogenous
stock solution.
substrate; and the chromogenic technique, based on the
development of colour after eleavage of a synthetic peptide- 4. Preparation of the standard endotoxin solutions
chromogen complexo
After vigorously mixing the standard endotoxin stock
The following 6 methods are described in the present solution, prepare appropriate serial dilutions of this solution
chapter: using water for BET.
Method A. Gel-elot method: limit test Use the solutions as soon as possible to avoid loss of activity
Method B. Gel-elot method: quantitative test by adsorption.
Method C. Turbidimetric kinetic method
Method D. Chromogenic kinetic method 5. Preparation ofthe test solutions
Method E. Chromogenic end-point method
Prepare the test solutions by dissolving or diluting active
Method F. Turbidimetric end-point method
substances or medicinal products using water for BET. Sorne
Proceed by any of the 6 methods for the test. In the event of substances or preparations may be more appropriately
doubt or dispute, the final decision is made based upon dissolved or diluted in other aqueous solutions. If necessary,
method A unless otherwise indicated in the monograph. adjust the pH of the test solution (or dilution thereof) so that
2014 Appendix XIV e V-A391
the pH of the mixture of the Iysate and test solution falls Mix a volume of the Iysate solution with an equal volume of
within the pH range specified by the Iysate manufacturer, 1 of the standard solutions (such as 0.1 mL aliquots) in each
usually 6.0 to 8.0. The pH may be adjusted by the use of tube. When single test vials or ampoules containing
acid, base or a suitable buffer, as recommended by the Iysate Iyophilised Iysate are employed, add solutions of standards
manufacturero Acids and bases may be prepared from directIy to the vial or ampoule. Incubate the reaction mixture
concentra tes or solids with water for BET in containers free for a constant period according to the recommendations of
of detectable endotoxin. Buffers must be validated to be free the Iysate manufacturer (usually at 37 ± 1 cC for
of detectable endotoxin and interfering factors. 60 ± 2 min), avoiding vibration. Test the integrity of the gel:
for tubes, take each tube in tum directly from the incubator
6. Determination ofthe Maximum Valid Dilution and invert it through approximately 180 in one smooth
0
The Maximum Valid Dilution (MVD) is the maximum motion. If a firm gel has formed that remains in place upon
allowable dilution of a sample at which .the endotoxin limit inversion, record the result as positive. A result is negative if
can be determined. Determine the MVD using the following an intact gel is not formed.
formulae: The test is considered valid when the lowest concentration of
endotoxin limit x concentration of test solution
the standard solutions shows a negative result in all replicate
MVD= tests.
The end-point is the lowest concentration in the series of
Endotoxin limit The endotoxin limit for active substances decreasing concentrations of standard endotoxin that clots
administered parenterally, defined on the basis of dose, is the Iysate. Determine the geometric mean end-point
equal to: concentration by ca1culting the mean of the logarithms of the
end-point concentrations of the 4 dilution series, take the
K antilogarithrn of this value, as indicated by the following
M expression:
K threshold pyrogenic dose of endotoxin per kilogram of

body mass, Geometric mean end-point concentration = antilog ~ e
M maximum recommended bolus dose of product per
kilogram of body mass.
Le sum of the log end-point concentrations of the
When the product is to be injected at frequent intervals or dilution series used,
infused continuously, M is the maximum total dose f number of replicates.
administered in a single hour periodo
The geometric mean end-point concentration is the
The endotoxin limit for active substances administered
measured sensitivity of the Iysate solution (IU/mL). If this is
parenterally is specified in units such as IU/mL, IU/mg,
not less than 0.5'A and not more than 2)" the labelled
IUlUnit of biological activity, etc., in monographs.
sensitivity is confirmed and is used in the tests performed
Concentration of test solution: with this Iysate.
- mglmL if the endotoxin limit is specified by mass (Il) TEST FOR INTERFERING FACTORS
(IU/mg),
Prepare solutions A, B, C and D as shown in
- Units/mL if the endotoxin limit is specified by unit of Table 2.6.14.-1 , and use the test solutions at a dilution less
biological activity (IUlUnit), than the MVD, not containing any detectable endotoxins,
- mUmL if the endotoxin limit is specified by volume operating as described under 1. Preparatory testing, (i)
(IU/mL). Confirmation of the labelled Iysate sensitivity.
'A = the labelled Iysate sensitivity in the gel-clot technique The geometric mean end-point concentrations of solutions B
(IU/mL) or the lowest concentration used in the standard and C are determined using the expression described in l.
curve of the turbidimetric or chromogenic techniques. Preparatory testing, (i) Confirmation of the labelled Iysate
sensitivity.
7. Gel-clot technique (Methods A and B) The test for interfering factors must be repeated when any
The gel-clot technique allows detection or quantification of changes are made to the experimental conditions that are
endotoxins and is based on clotting of the Iysate in the likely to influence the result of the test.
presence of endotoxins. The minimum concentration of The test is considered valid when all replica tes of solutions A
endotoxins required to cause the Iysate to clot under and D show no reaction and the result of solution C
standard conditions is the labelled Iysate sensitivity. confirms the labelled Iysate sensitivity.
To ensure both the precision and validity of the test, confirm If the sensitivity of the Iysate determined with solution B is
the labelled Iysate sensitivity and perform the test for not less than 0.5), and not greater than 2'A, the test solution
interfering factors as described under l. Preparatory testing. does not contain interfering factors under the experimental
1. Preparatory testing conditions used. Otherwise, the test solution interferes with
(1) CONFIRMATION OF THE LABELLED LYSATE SENSITIVITY the test.
Confirm in 4 replica tes the labelled sensitivity 'A, expressed in If rhe preparation being examined interferes with the test at a
IU/mL, of the Iysate solution prior to use in the test. dilution less than the MVD, repeat the test for interfering
Confirmation of the Iysate sensitivity is carried out when a factors using a greater dilution, not exceeding the MVD .
new lot of Iysate is used or when there is any change in the The use of a more sensitive Iysate permits a greater dilution
test conditions which may affect the outcome of the test. of the preparation being examined and this may contribute to
Prepare standard solutions of at least 4 concentrations the elimination of interference.
equivalent to 2'A, le, 0.5'A and 0.25'A by diluting the standard Interference may be overcome by suitable validated
endotoxin stock solution with water for BET. treatment, such as filtration, neutralisation, dialysis or heat
V-A392 Appendix XIV e 2014
Table 2.6.14.-1
Solution Endotoxin concentration/Solution to which Diluent Dilution factor Endotoxin Number oC replicates
endotoxin is added concentration
A None/ Test solution 4
B 2A.;Test solution Test solution 1 2A 4

2 11.. 4
4 0.51.. 4
8 0.251.. 4
e 21../Water for BET Water for BET 2A 2
2 lA. 2
4 0.51.. 2
8 0.251.. 2
D None/Water for BET 2
Solution A = solution of the preparation being examined that is free of detectable endotoxins.
Solution B = test for interference.
Solution e = control of the labelled Iysate sensitivity.
Solution D = negative control (water for BET).
treatment. To establish that the treatment chosen effectively However, if the preparation does not comply with the test at
elimina tes interference without loss of endotoxins, repeat the a dilution less than the MVD, the test may be repeated using
test for interfering factors using the preparation being a greater dilution, not exceeding the MVD.
examined to which the standard endotoxin has been added 3. Quantitative test (Method B)
and which has then been submitted to the chosen treatment.
(1) PROCEDURE
2. Limit test (method a) The test quantifies bacterial endotoxins in the test solution
(1) PROCEDURE by titration to an end-point. Prepare solutions A, B, C and D
Prepare solutions A, B, C and D as shown in as shown in Table 2.6.14.-3, and test these solutions
Table 2.6.14.-2, and perform the test on these solutions according to the procedure described under l . Preparatory
following the procedure described under 1. Preparatory testing, (i) Confirmation of the labelled Iysate sensitivity.
testing, (i) Confirmation of the labelled Iysate sensitivity. (1I) CALCULATION AND INTERPRETATION
The test is considered valid when the following 3 conditions
Table 2.6.14.-2 are met:
Solution Endotoxin concentration/ Solution Number oC replicates (a) both replicates of solution D (negative control) are
to which endotoxin is added
A
negarive,
None/ Diluted test solution 2
(b) both replicates of solution B (positive product control)
B 2A.;Diluted test solution 2
are positive,
e 2A/Water for BET (c) the geometric mean end-point concentration of solution
D None/Water for BET 2 C is in the range of O.5A to 2A.
To determine the endotoxin concentration of solution A,
calculate the end-point concentration for each replica te, by
Prepare solution A and solution B (positive product control) multiplying each end-point dilution factor by A.
using a dilution not greater than the MVD and treatments as
described in l. Preparatory testing, (ii) Test for interfering The endotoxin concentration in the test solution is the
factors. Solutions B and C (positive controls) contain the end-point concentration of the replica tes. If the test is
standard endotoxin at a concentration corresponding to twice conducted with a diluted test solution, calculate the
the labelled Iysate sensitivity. Solution D (negative control) concentration of endotoxin in the original solution by
consists of water for BET. multiplying the result by the dilution factor.
If none of the dilutions of the test solution is positive in a
(1I) INTERPRETATION
valid test, report the endotoxin concentration as les s than A
The test is considered valid when both replica tes of
(or, if a diluted sample was tested, report as less than the
solution B and C are positive and those of solution D are
10west dilution factor of the sample x A) . If all dilutions are
negative.
positive, the endotoxin concentration is reported as equal to
When a negative result is found for both replica tes of or greater than the largest dilution factor multiplied by A
solution A, the preparation being examined complies with the (e.g. in Table 2.6.14.-3, the inirial dilution factor x 8 x A) .
test.
The preparation being examined meets the requirements of
When a positive result is found for both replicates of solution the test if the endotoxin concentration in both replica tes is
A, the preparation being examined does not comply with the les s than that specified in the monograph.
test.
When a positive result is found for one replicate of solution 8. Photometric quantitative techniques (Methods e,
A and a negative result is found for the other, repeat the test. D, E and F)
In the repeat test, the preparation being examined complies 1. Turbidimetric technique (Methods e and F)
with the test if a negative result is found for both replicates of This technique is a photometric test to measure the increase
solution A. The preparation does not comply with the test if in turbidity. Based on the test principie employed, this
a positive result is found for one or both replica tes of technique may be classified as being either the end-point-
solution A. turbidimetric test or the kinetic-mrbidimetric test.
2014 Appendix XIV e V-A393
rabIe 2.6.14.-3
Solution Endoloxin concenlratíon/Solulion lo which Diluenl Dilution faclor Endotoxin Number of replicales
endoloxin is added concenlration
A None/ Test solution Water for BET 2
2 2
4 2
8 2
B 2A;Tesl solution 2/- 2
e 2A/Water for BET Water for BET 2A 2

2 lA 2
4 0.51.. 2
8 0.251.. 2
D None/Water for BET 2
Solution A = test solution al the dilution, not exceeding the MVD, with which the test for interfering factors was carried out. Subsequent dilution of the
test solution must not exceed the MVD . Use water for BET to make a dilution series of 4 tubes containing the test solution at concentrations of 1, 1/2,
1/4 and 1/8, relative to the dilution used in the test for interfering factors. Other dilutions up to the MVD may be used as appropriate.
Solution B = solution A containing standard endotoxin at a concentration of 2A (positive product control).
Solution e = a dilution series of 4 tubes of water for BET containing the standard endotoxin at concentrations of n, A, 0.51.. and 0.25/-.
Solution D = water for BET (negative control).
The end-point-turbidimetric test (Method F) is based on the solution as recommended by the lysate manufacturer (volume
quantitative relationship between the endotoxin concentration ratios, incubation time, temperature, pH, etc.).
and the turbidity (absorbance or transmission) of the reaction If the desired range is greater than 2 log in the kinetic
mixture at the end of an incubation periodo methods, additional standards must be included to bracket
The kinetic-turbidimetric test (Method C) is a method to each log increase in the range of the standard curve .
measure either the time (onset time) needed for the reaction The absolute value of the correlation coefficient, I r 1, must
mixture to reach a predetermined absorbance or be greater than or equal to 0.980, for the range of endotoxin
transmission, or the rate of turbidity development. concentrations set up.
The test is carried out at the incubation temperature (JI) TEST FOR INTERFERING FACTORS
recommended by the Iysate manufacturer (usualIy Selecr an endotoxin concentration at or near the middle of
37 ± 1 °C).
the endotoxin standard curve.
2. Chromogenic technique (Methods D and E) Prepare solutions A, B, e and D as shown in
This technique is used to measure the chromophore relea sed Table 2.6.14.-4. Perform the test on at least 2 replica tes of
from a suitable chromogenic peptide by the reaction of these solutions as recommended by the Iysate manufacturer
endotoxins with the lysate. Depending on the test principIe (volume of test solution and Iysate solution, volume ratio of
employed, this technique may be classified as being either the test solution 10 lysate solution, incubation time, etc.).
end-point-chromogenic test or the kinetic-chromogenic test.
The end-point-chromogenic test (Method E) is based on the Table 2.6.14.-4.
quantitative relationship between the endotoxin concentration Solutíon Endoloxin Solutíon lo which Number oC
and the quantity of chromophore released at the end of an concenlration endotoxin is added rel!licates
incubation periodo A None Test solution Not less than 2
The kinetic-chromogenic test (Method D) measures either B Middle concentration of Test solution Not less than 2
the time (onset time) needed for the reaction mixture 10 the standard curve
reach a predetermined absorbance, or the rate of colour. e At least3 concentrations Water for BET Each concentration
(Iowest concentration not less than 2
development. is designated Al
The test is carried out at the incubation temperature D None Water for BET Not less than 2
recommended by the lysate manufacturer (usualIy Solution A = test solution, that may be diluted not to exceed the MVD.
37 ± 1 oC). Solution B = preparation to be examined at the same dilution as solution A,
containing added endotoxin at a concentration equal to or near the middle
3. Preparatory testing of the standard curve.
To assure the precision or validity of the turbidimetric and Solution e = standard endotoxin solution at the concentrations used in
the validation of the method as described under 3. Preparatory testing,
chromogenic techniques, preparatory tests are conducted 10 (i) Assurance of criteria for the standard curve (positive controls).
show that the criteria for the standard curve are satisfied and Solution D = water for BET (negative control).
that the test solution does not interfere with the test.
Validation of the test method is required when any changes
The test is considered va lid when the folIowing conditions
are made to the experimental conditions that are likely to
are met:
inftuence the result of the test.
- the absolute value of the correlation coefficient of the
(1) ASSURANCE OF CRITERlA FOR THE STANDARD CURVE
standard curve generated using solution e is greater than
The test must be carried out for each lor of lysate reagent. or equal 10 0.980;
Using the standard endotoxin solution, prepare at least - the result with solution D does not exceed the limit of the
3 endotoxin con centra tion s within the range indicated by the blank value required in the description of the Iysate
Iysate manufacturer 10 generate the standard curve. Perform reagent employed, or it is less than the endotoxin
the test using at least 3 replica tes of each standard endotoxin detection limit of the lysate reagent employed.
V-A394 Appendix XIV D 2014
Calculate the mean recovery of the added endotoxin by week preceding the test. A rabbit is not be used in a pyrogen
subtracting the mean endotoxin concentration in the solution test:
(if any) (solution A, Table 2.6.14.-4) from that in the a) if it has been used in a negative pyrogen test in the
solution containing the added endotoxin (solution B, preceding 3 days, or
Table 2.6.14 .-4). b) if it has been used in the preceding 3 weeks in a pyrogen
The test solution is considered free of interfering factors if test in which the substance under examination failed to
under the conditions of the test, the measured concentration pass the test.
of the endotoxin added to the test solution is within Animals' quarters Keep the rabbits individually in a
50-200 per cent of the known added endotoxin quiet area with a uniforrn appropriate temperature. Withhold
concentration, after subtraction of any endotoxin detected in food from the rabbits ovemight and until the test is
the solution without added endotoxin. completed; withhold water during the test. Carry out the test
When the endotoxin recovery is out of the specified range, in a quiet room where there is no risk of disturbance exciting
the test solution is considered to Contain interfering factors. the animals and in which the room temperature is within
Repeat the test using a greater dilution, not exceeding the 3 oC of that of the rabbits' living quarters, or in which the
MVD. Furthermore, interference of the test solution or rabbits have been kept for at least 18 h before the test.
diluted test solution not to exceed the MVD may be Materials Glassware syringes and needles. Thoroughly
eliminated by suitable validated treatment, such as filtration, wash all glassware, syringes and needles with water for
neutralisation, dialysis or heat treatment. To establish that injections and heat in a hot-air oven at 250 oC for 30 min or
the treatment chosen effectively elimina tes interference at 200 oC for 1 h.
without loss of endotoxins, repeat the test for interfering
Retaining boxes The retaining boxes for rabbits whose
factors using the preparation being examined to which the
temperature is being measured by an electrical device are
standard endotoxin has been added and which has then been
made in such a way that the animals are retained only by
submitted to the chosen treatment.
loosely fitting neck-stocks; the rest of the body remains
4. Test relatively free so that the rabbits may sit in a normal
(1) PROCEDURE position. They are not restrained by straps or other similar
Follow the procedure described in 3. Preparatory testing, (ii) methods which may harm the animal. The animals are put
Test for interfering factors. into the boxes not less than 1 h before the first record of the
(U) CALCULATION temperature and remain in them throughout the test.
Calculate the endotoxin concentration of each replicate of Thermometers Use a therrnometer or electrical device
solution A using the standard curve generated by the positive which indicates the temperature with a precision of 0.1 oC
control solution e. and insert into the rectum of the rabbit to a depth of about
The test is considered valid when the following 3 5 cm. The depth of insertion is constant for any one rabbit
requirements are met: in any one test. When an electrical device is used it may be
left in position throughout the test.
(1) the results obtained with solution C comply with the
requirements for validation defined under 3. Preparatory Preliminary test
testing, (i) Assurance of criteria for the standard curve,
After selection of the animals, one to three days before
(2) the endotoxin recovery, calculated from the endotoxin testing the product to be examined, treat those animal s that
concentration found in solution B after subtracting the have not been used during the previous 2 weeks by
endotoxin concentration found in solution A, is within the intravenous injection of 10 mL per kilogram of body mass of
range of 50-200 per cent, a pyrogen-free 9 giL s~lution of sodium ehloride R warmed to
(3) the result obtained with solution D (negative control) about 38.5 0e. Record the temperatures of the animals,
does not exceed the limit of the blank value required in the beginning at least 90 min before injection and continuing for
description of the lysate employed, or it is les s than the 3 h after the injection of the solution. Any animal showing a
endotoxin detection limit of the lysate reagent employed. ' temperature variation greater than 0.6 oC is not used in the
(m ) INTERPRETATION main test.
The preparation being examined complies with the test if the
mean endotoxin concentration of the replicates of solution A, Main test
after correction for dilution and concentration, is less than Carry out the test using a group of three rabbits.
the endotoxin limit for the product. Preparation and injection 01 the product Warm the
Guidelines on the test lor bacterial endotoxins are given in general liquid to be examined to approximately 38,5 oC before the
ehapter 5.1. JO. injection. The product to be examined may be dissolved in,
or diluted with, a pyrogen-free 9 gIL solution of sodium
ehloride R or another prescribed liquido Inject the solution
slowly into the marginal vein of the ear of each rabbit over a
D. Test tor Pyrogens períod not exceeding 4 min, unless otherwise prescribed in
(Ph. Eur. method 2.6.8) the monograph. The amount of the product to be injected
The test consists of measuring the rise in body temperature varíes according to the product to be examined and is
evoked in rabbits by the intravenous injection of a sterile prescribed in the monograph. The volume injected is not less
solution of the substance to be examined. than 0.5 mL per kilogram and not more than 10 mL per
Selection of animals Determination 01 the initial and maximum
Use healthy, adult rabbits of either sex weighing not les s than temperatures The "initial temperature" of each rabbit is
1.5 kg, fed a complete and balanced diet not containing the mean of two temperature readings recorded for that
antibiotics, and not showing loss of body mass during the rabbit at an interval of 30 min in the 40 min immediately
2014 Appendix XIV F V-A395
preceding the injection of the product to be examined. label of the preparation to be examined or on the
The "maximum temperature" of each rabbit is the highest accompanying leaflet. Observe the animals for 7 days .
temperature recorded for that rabbit in the 3 h after the The preparation passes the test if none of the animals shows
injection. Record the temperature of each rabbit at intervals signs of ill health. If more than one animal dies, the
of not more than 30 min, beginning at least 90 min before preparation fails the test. If one of the animals dies or shows
the injection of the product to be examined and continuing signs of ill health, repeat the test. The preparation passes the
3 h after the injection. The difference between the maximum test if none of the animals in the 2nd group die or shows
temperature and the initial temperature of each rabbit is signs of ill health in the time interval specified.
taken to be its response. When this difference is negative, the
The test must also be carried out on 2 healthy guinea-pigs
result is counted as a zero response.
weighing 250 g to 400 g. Inject intraperironeally into each
Rabbits showing a temperature variation greater than 0.2 oC animal 1 human dose but not more than 5 .0 mL.
between two successive readings in the determination of the The human dose is that stated on the label of the preparation
initial temperature are withdrawn from the test. In any one to be examined or on the accompanying leaflet. Observe the
test, only rabbits having initial temperatures which do not animals for 7 days.
differ from one another by more than 1 oC are used.
The preparation passes the test if none of the animals shows
All rabbits having an initial temperature higher than 39.8 oC
signs of ill health. If more than one animal dies the
or less than 38.0 cC are withdrawn from the test.
preparation fails the test. If one of the animals dies or shows
Interpretation 01 results Having carried out the test first signs of ill health, repeat the test. The preparation passes the
on a group of three rabbits, repeat if necessary on further test if none of the animals in the 2nd group die or shows
groups of three rabbits to a total of four groups, depending signs of ill health in the time interval specified.
on the results obtained. If the summed response of the first
group does not exceed the figure given in the second column
of the Table 2.6 .8.-1, the substance passes the test. If the
summed response exceeds the figure given in the second F. Test for Depressor Substances
column of the table but do es not exceed the figure given in (Ph. Eur. methad 2.6.11)
the third column of the table, repeat the test as indicated Carry out the test on a cat weighing not less than 2 kg and
aboye. If the surnmed response exceeds the figure given in anaesthetised with chloralose or with a barbiturate that allows
the third column of the table, the product fails the test. the maintenance of uniform blood pressure. Protect the
animal from loss of body heat and maintain it so that the
Table 2.6.8.-1 rectal temperature remains within physiologicallimits.
Number of rabbits Product passes if summed Product fails if summed Introduce a cannula into the trachea. Insert a cannula filled
resJ20nse does not exceed resJ20nse exceeds
with a heparinised 9 gIL solution of sodium chloride into the
3 1.15 oC 2.65 oC
common carotid artery and connect it ro a device capable of
6 2.80 oC 4.30 oC giving a continuous record of the blood pressure. Insert into
4.45 oC 5.95 oC the femoral vein another cannula, filled with a heparinised
9 giL solution of sodium chloride, through which can be
12 6.60 oC 6.60 oC
injected the solutions of histamine and of the substance ro be
examined. Determine the sensitivity of the animal to
Rabbits used in a test for pyrogens where the mean rise in histamine by injecting intravenously at regular intervals, doses
the rabbits' temperature has exceeded 1.2 oC are of histamine solunan R corresponding ro 0.1 /lg and 0.15 /lg of
permanently excluded. histamine base per kilogram of body mass. Repeat the lower
dose at least 3 times. Administer the second and subsequent
injections not less than 1 min after the blood pressure has
returned to the leve! it was at immediately before the
E. Test for Abnormal Toxicity previous injection. The animal is used for the test only if a
readily discernible decrease in blood pressure that is constant
(Ph. Eur. mechad 2.6.9) for the lower dose is obtained and if the higher dose causes
greater responses. Dissolve the substance to be examined in
General test sufficient of a 9 gIL solution of sodium chloride or other
Inject intravenously into each of 5 healthy mice, weighing prescribed solvent, to give the prescribed concentration.
17 g to 24 g, the quantity of the substance to be examined Inject intravenously per kilogram of body mass 1.0 mL of
prescribed in the monograph, dissolved in 0.5 mL of water fa r histamine solution R, followed by 2 successive injections of the
injecnans R or of a 9 gIL sterile solution of sadium chlaride R. prescribed amount of the solution to be examined and,
Inject the solution over a period of 15 s to 30 s, unless finally, 1.0 mL of histamine salurion R. The second, third and
otherwise prescribed. fourth injections are given not less than 1 min after the blood
The substance passes the test if non e of the mice die within pressure has returned to the level it was at immediately
24 h or within such time as is specified in the individual before the preceding injection. Repeat this series of injections
monograph. If more than one animal dies the preparation twice and conclude the test by giving 1.5 mL of histamine
fails the test. If one of the animals dies, repeat the test. salution R per kilogram of body mass.
The substance passes the test if non e of the animals in the If the response ro 1.5 mL of histamine solution R per kilogram
2nd group die within the time interval specified. of body mass is not greater than that to 1.0 mL the test is
invalid o The substance to be examined fails the test if the
Irnrnunosera (antisera) and vaccines mean of the series of responses to the substance is greater
Unless otherwise prescribed, inject intraperitoneally 1 human than the mean of the responses to 1.0 mL of histamine
dose but not more than 1.0 mL into each of 5 healthy mice, salution R per kilogram of body mass or if any one dose of
weighing 17 g to 24 g. The human dos e is that stated on the the substance causes a greater depressor response than the
V-A396 Appendix XIV G 2014
concluding dose of the histamine solution. The test animal invalid and the test for depressor substances (2.6. 11) must be
must not be used in another test for depressor substances if carried out.
the second criterion applies or if the response to the high
dose of histamine given after the administration of the Solution A
substance ro be examined is less than the mean response to Sodium chloride 160.0 g
the low doses of histamine previously injected. Potassium chloride 4.0 g
Calcium chloride, anhydrous 2.0 g
Magnesium chloride, anhydrous 1.0g
Disodium hydrogen phosphate dodecahydrate 0.10 g
G. Test for Histamine Water far injectians R ro 1000mL
(Ph. Eur. methad 2.6.10) Solution B
Euthanise a guinea-pig weighing 250 g ro 350 g that has
SolutionA 50.0 mL
been deprived of food for the preceding 24 h. Remove a
Atropine sulfate 0.5 mg
portion of the distal small intestine 2 cm in lengrh and empry
Sodium hydrogen carbonate 1.0 g
the isolated part by rinsing carefully with solution B
Glucose monohydrate 0.5 g
described below using a syringe. Attach a fine thread to each
Water far injectians R ro 1000 mL
end and make a small transverse incision in the middle of the
pie ce of intestine. Place it in an organ bath with a capaciry of Solution B should be freshly prepared and used within 24 h.
10 mL to 20 mL, containing solution B maintained at a
constant temperature (34 oC to 36 oC) and pass through the
solution a current of a mixture of 95 parts of oxygen and
5 parts of carbon dioxide. Attach one of the threads near ro H. Monocyte-Activation Test
the botrom of the organ bath. Attach the other thread to an (Ph. Eur. methad 2.6.30)
isoronic myograph and record the contractions of the organ
on a kymograph or other suitable means of giving a 1 Introduction
permanent record. If a lever is used, its length is such that The monocyte activation test (MAT) is used ro detect or
the movements of the organ are amplified about 20 times. quantify substances that activate human monocytes or
The tension on the intestine should be about 9.8 mN (1 g) monocytic cells ro release endogenous mediators: such as
and it should be adjusted to the sensitiviry of the organ. pro-infiammarory cytokines, for example tumour necrosis
Flush out the organ bath with solution B. Allow it to stand factor alpha (TNFa), interJeukin-1 beta (IL-1~) and
for 10 minoFlush 2 or 3 times more with solution B. interleukin-6 (IL-6) . These cytokines have a role in fever
Stimulate a series of contractions by the addition of pathogenesis. Consequently, the MAT will detect the
measured volumes between 0.2 mL and 0.5 mL of a solution presence of pyrogens in the test sample. The MAT is
of histamine dihydrachlaride R having a strength which suitable, after a product-specific validation, as a replacement
produces reproducible submaximal responses. This dose is for the rabbit pyrogen test.
termed the "high dose". Flush the organ bath (preferably by Pharmaceutical products that contain non-endotoxin
overflow without emprying the bath) 3 times with solution B pyrogenic or pro-infiammatory contaminants often show very
before each addition of histamine. The successive additions steep dose-response curves in comparison with endotoxin
should be made at regular intervals allowing a complete dose-response curves. Frequently the greatest response ro
relaxation between additions (about 2 min). Add equal such contaminated products is obtained with undiluted
volumes of a weaker dilution of histamine dihydrachlaride R solutions of the preparations being examined or small
which produces reproducible responses approximately half as dilutions of the preparations being examined. For this reason
great as the "high dose". This dose is termed the "low preparations that contain or may contain non-endoroxin
dos e" . Continue the regular additions of "high" and "low" contaminants have ro be tested at a range of dilutions that
doses of histamine solution as indicated aboye, and alternate includes minimum dilution.
each addition with an equal volume of a dilution of the
The following 3 methods are described in the present
solution ro be examined, adjusting the dilution so that the
chapter.
contraction of the intestine, if any, is smaller than that due to
the "high dose" of histamine. Determine whether the Method A. Quantitative test
contraction, if any, is reproducible and that the responses ro Method B. Semi-quantitative test
the "high" and "low" doses of histamine are unchanged. Method C. Reference lot comparison test
Calculate the activiry of the substance to be examined in The test is carried out in a manner that avoids pyrogen
terms of its equivalent in micrograms of histamine base from contamination.
the dilution determined as aboye.
The quantiry so determined do es not exceed the quantity 2 Definitions
prescribed in the monograph. The maximum valid dilution (MVD) is the maximum
If the solution to be examined do es not produce a allowable dilution of a sample at which the contaminant limit
contraction, prepare a fresh solution adding a quantiry of can be determined. Determine the MVD using the following
histamine corresponding to the maximum tolerated in the expression:
monograph and note whether the contractions produced by
the preparation with the added histamine correspond to the CLCxC
amount of histamine added. If this is not the case, or if the LOD
contractions caused by the substance to be examined are not
reproducible or if subsequent responses to "high" and "low" eLe contaminant limit concentration;
doses of histamine are diminished, the results of the tests are e concentration of test solution;
LOD limit of detection.
2014 Appendix XIV H V-A397
The acceptance criterion for a pass/fail decision is the medium, e.g. 5 per cent CO 2 in humidified air. The duration
contaminant limit concentration (CLC), which is expressed of the culture is sufficient to allow accumulation of the
in endotoxin equivalents per milligram or millilitre, or in chosen read-out. The responses of the chosen read-out,
units of biological activity of the preparation being examined. e.g. a pro-inftammatory or pyrogenic cytokine, to a solution
The CLC is calculated using the fo llowing expression: of the preparation being examined are compared with
responses to standard endotoxin or to a reference lot of the
K preparation being examined. The chosen read-out method is
M calibrated using the appropriate standard.
K threshold pyrogenic dose of endotoxin per kilogram of 4 Apparatus

body mass; Depyrogenate all glassware and O1her heat-stable apparatus in
M maximum recommended bolus dose of product per a hot-air oven using a validated process. A commonly used
kilogram of body mass. minimum time and temperature is 30 min at 250 oc.
If employing plastic apparatus, such as microtitre plates and
When the product is to be injected at frequent intervals or
infused continuously, M is the maximum total dose pipette tips for automatic pipetters, use apparatus shown 10
administered in a single hour periodo be free of detectable pyrogens and which do not interfere
with the test.
Where an endotoxin limit concentration (ELC) has been
specified for a product, the CLC is the same as the ELC, 5 Cell Sources and Qualification
unIess otherwise prescribed. In this case, the concentration of
5-1 Whole blood
test solution is expressed in mglmL if the endotoxin limit is
specified by mass (IU/mg), in Units/mL if the endotoxin Whole blood is obtained from single donors or from pooled
limit is specified by unit of biological activity (IUlUnit), in whole blood which are qualified according to the
mUmL if the endotoxin limit is specified by volume requirements described under section 5-3 and section 5-4,
(IU/mL). respectively.
Endotoxin equivalents are values for the contaminant 5-2 Peripheral blood mononuclear cells (PBMC)
concentration read off the standard endotoxin dose-response PBMC are isolated from blood obtained from single donors
curve (Method A) or estimated by comparison with or from pooled whole blood which are qualified according to
responses to standard endotoxin solutions (Method B). the requirements described under section 5-3 and section
The standard endotoxin stock solution is prepared from an 5-4, respectively.
endotoxin reference standard that has been calibrated against
5-3 Qualification 01 blood donors
the International Standard, for example endotoxin
standard BRP. Blood donors are to satisfY the following qualification criteria,
together with other requirements in force that relate to
The cut-off value is calculated using the following expression:
consent, health and safety and ethical considerations. Blood
donors pre ro describe themselves as being in good health, as
x+ 38 not ro be suffering from any bacterial or viral infections and
ro have been free from the symptoms of any such infection
X mean of the 4 replica tes for the responses to the blank
for a period of at least 1 week prior to the donation of blood.
(Ro);
Blood donors are n01 to have taken non-steroidal anti-
standard deviation of the 4 replica tes of the responses
infiammatory drugs during the 48 h prior to donating bIood
to the blank (Ro).
and steroidal anti-inftammarory drugs during the 7 days prior
The cut-off value is expressed in units appropriate to the to donating blood. Individuals who have been prescribed
read-out. immunosuppressant or other drugs known to inftuence the
The limit of detection (LOD) is determined using the production of the chosen readout are not to serve as blood
endotoxin standard curve . The LOD is the concentration of donors. Blood donation are to be tested for infection markers
endotoxin corresponding to the cut-off value . For the according to national requirements for transfusion medicine.
purpose of the test, the LOD is expressed as endotoxin 5-4 Qualification 01 cells pooled Irom a number 01
equivalents per millilitre. donors
Pools (of whole blood or blood fractions, e.g. PBMC), must
3 General procedure
consist of donations from a minimum of 4 individual donors
A solution of the preparatión b,eing examined is incubated but preferably 8 or more donors, where practicable, taking
with a source of human monocytes or human monocytic from each donation an approximately equal volume of blood,
cells, e.g. from human heparinised peripheral blood that is or cells from an approximately equal volume of blood.
preferably not more than 4 h old, or a monocyte-containing For the qualification of pooIed cells proceed as follows:
fraction of that blood, such as human peripheral blood within 4 hours of collection of blood, generate dos e-response
mononuc1ear cells (PBMC) isolated, e.g. by density-gradient curves from the pool using standard endotoxin with at least
centrifugation, or a human monocytic cell line. Human 4 geometrically diluted endotoxin concentrations, e.g. in the
heparinised peripheral blood is usually diluted with culture range of 0.01 IU/mL to 4 IU/mL. The dose-response curves
medium or saline e.g. to 2-50 per cent V/V (final are to meet the 2 criteria for the standard curve described
concentration). PBMC or monocytic cell lines, in culture under section 6-1.
medium and with either the donor's own plasma or AB
serum, are typically used at a final cell density of O.1-1. O x 5-5 Qualification 01 cryo-preserved cells
10 6 cells per well, tube or other receptacle. For monocytic The cell source intended for use in a MAT, e.g. human
celllines, heat-inactivated foetal bovine serum may be whole blood, blood fractions, such as PBMC or monocytic
substituted for AB serum. The cell culture is carried out at cell lines, may be cryo-preserved. Pools of cryo-preserved
37 ± 1 °C, in an atmosphere appropriate for the culture cells are obtained by pooling before freezing, or by pooling
V-A398 Appendix XIV H 2014
single cryo-preserved donations irnmediately after thawing. same dilutions of the preparation to be examined and add
Pools must consist of donations from a minimum of 4 endotoxin at a concentration equal to or near the middle of
individual donors but preferably 8 or more donors where the standard curve (Method A) or equal to twice the LOD
practicable, taking from each donation an approximately (Method B), or use a diluent containing added endotoxin at
equal volume of blood, or cells from an approximately equal a concentration equal to or near the middle of the standard
volume of blood. Qualification of cryo-preserved blood or curve (Method A) or equal to twice the LOD (Method B).
cells is performed immediately after thawing (and pooling if Test these dilution series in paraUel in the same experimento
necessary): dose-response curves for cryo-preserved blood or Use the standard curve to calculate the concentration of
cells are to comply with the 2 criteria for the standard curve endotoxin-equivalents in each solution. Calculate the mean
as described under section 6-1. recovery of the added endotoxin by subtracting the mean
5-6 Monocytic continuous celllines concentration of endotoxin equivalents in the solution (if
any) from that in the solution containing the added
A human monocytic cell line is continuously cultured in
endotoxin. The test solution is considered free of interfering
order to warrant a sufficient supply for the MAT.
factors if, under the conditions of the test, the measured
To optimise the method, clones derived from the cell line
endotoxin equivalents in the test solution to which endotoxin
can be used.
is added is within 50-200 per cent of the added
Cells must be maintained under aseptic conditions and concentration, after subtraction of any endotoxin equivalents
regularly tested for the presence of mycoplasma detected in the solution without added endotoxin. When this
contamination. Additionally, cells must be regularly checked criterion is not met, Method C is to be preferred over
for identity (e.g. doubling time, morphology, and function) Methods A and B.
and stability. The functional stability of a cell line is assessed
In Method C, a solution of the preparation being examined
by monitoring its performance in relation to the number of
is tested at 3 dilutions: the highest concentration (lowest
passages during routine testing. Criteria for functional
dilution) that stimulates the greatest release of the chosen
stability are to be established and may include growth
read-out and the 2-fold dilutions immediately below and
criteria, maximum signal obtained in the test, background
aboye the chosen dilution. Since the concentration that
noise and receptor expression. The receptor expression may
stimulates the greatest release of the chosen read-out may be
be tested with specific ligands e.g. lipopolysaccharide (LPS)
donor-dependent as well as batch-dependent, the product-
for toll-like receptor 4 (TLR4), lipoteichoic acid (LTA) for
specific validation is to be performed in at least 3
toll-like receptor 2 (TLR2), synthetic bacterial lipoprotein for
independent tests, each using ceUs from different donors.
TLR2-TLR1 or synthetic bacterial lipoprotein for TLR2-
The highest concentration (lowest dilution) that stimulates
TLR6.
the greatest release of the chosen read-out in the majority of
donors, and the 2-fold dilutions immediately below and
6 Preparatory testing
aboye that dilution are deemed to be validated for further
To ensure both the precision and validity of the test, testing. If undiluted test solution stimulates the greatest
preparatory tests are conducted, to assure that the criteria for release of the chosen read-out, subsequent testing is to be
the standard curve are satisfied, that the solution does not . performed using undiluted test solution and also test solution
interfere with the test, tha t the test detects endotoxins and diluted in the ratios 1:2 and 1:4 before its addition to the
non-endotoxins contaminants and that the solution does not PBMe. The 3 dilutions to be used in subsequent testing are
interfere in the detection system. not to exceed the MVD; the dilution factors for these 3
6-1 Assurance of criteriafor the standard curve solutions are designated 110 12 and 13' Following the product-
Using the standard endotoxin solution, prepare at least 4 specific validation, the test is routinely performed with cells
endotoxin concentrations to generate the standard curve. from 4 individual donors or a single pool or with cells from
Perform the test using at least 4 replicates of ea eh 1 passage of a human monocytic cell lineo
concentration of standard endotoxin. 6-3 Method validation for non-endotoxin monocyte-
The basal release of the chosen read-out (blank) in the activating contaminants
absence of added standard endotoxin is to be optimised to be The preparatory testing is also to show that the chosen test
as low as possible. system detects, in addition to bacterial endotoxins, non-
There are 2 acceptance criteria for the standard curve: endotoxin pro-inflammatory or pyrogenic contaminants. This
- the regression of responses (appropriately transformed if can be achieved using historie batches that have been found
necessary) on log dose shall be statistically significant (p < to be contaminated with non-endotoxin contaminants that
0.01); caused positive responses in the rabbit pyrogens test or
adverse drug reactions in mano Where such batches are not
- the regression of responses on log dose must not deviate
available, the preparatory testing is to include validation of
significantly from linearity (p > 0.05). If analysis for a
the test system using specific ligands for toll-like receptors,
4-parameter logistic curve is performed, then the observed
e.g. peptidoglycans, lipoteichoic acids or synthetic bacterial
curve must not deviate significantly from the theoretical
lipoproteins.
curve as calculated by using the usual statistical methods
(see chapter 5.3. Statistical analysis). 6-4 Interference in the detection system
6-2 Testfor interfering factors Once the optimum dilution of the solution of the preparation
being examined for further testing has been identified, this
To assure the validity of the test, preparatory tests are
dilution is tested for interference in the detection system
conducted to assure that the test solution does not interfere
(e.g. ELISA) for the chosen read-out. The agreement
with the test. Validation of the test method is required when
between a dilution series of the standard for the chosen read-
any changes are made to the experimental conditions that are
out, in the presence and absence of the test preparation, is to
likely to influence the result of the test. U sing an appropriate
be within ± 20 per cent.
diluent, dilute the preparation to be examined in geometric
steps, with all dilutions not exceeding the MVD. Make the
2014 Appendix XIV H V-A399
7 Methods (1 donor exc1uded for failing ro comply with test

7-1 Method A: quantitative test performance criteria or showing a positive reaction), the test
is continued with cells from a further 4 donors, none of
Method A involves a comparison of the preparation being
whom provided cells for the 1sr test, and the preparation
examined with a standard endotoxin dose-response curve.
being examined is required to pass the test with cells from 7
T he contaminant concentration of the prepararion being
of the 8 different donors (i.e. a maximum of 1 positive
examined is ro be less than the eLe ro pass rhe test.
reaction in 8 donors is allowed). When the source of
7-1-1 Test pracedure monocytes consists of cells pooled from a number of
Using the validated test method, prepare the solutions shown individual donors, the preparation being examined is required
in Table 2.6.30.-1 and culture 4 replicates of each solution ro pass the test with 1 pool of cells. Where a human
with cells from each of 4 individual donors or a single pool monocytic cell line is used for the test, the preparation being
or with cells from 1 passage of a human monocytic cell line. examined is required to pass the test with I passage of the
cellline.
Table 2.6.30.-1
7-2 Method B: semi-quantitative test
Solution Solution
Added endotoxin Number of Method B involves a comparison of the preparation being
(IV/mL) replicates
examined with standard endotoxin. The contaminant
A Test solution/ f None 4 concentration of the test preparation it to be less than the
B Test solution/2 x f None 4 eLe to pass the test. Solution A must be chosen for the
release decision, unless otherwise justified and authorised.
e Test solution/ 4 x f None 4
7-2-1 Test praeedure
Middle dose
from endotoxin Using the validated test method, prepare the solutions shown
D Test solution/ f 4
standard curve in Table 2.6.30.-2 and culture 4 replicates of each solution
(R)
with cells from each of 4 individual donors or a single pool
Pyrogen-free saline None (negative or with cells from 1 passage of a human monocyric cell line.
Ro 4
or test diluent control)
4 concentrations
Pyrogen-free saline 4 of each
R,-R. or test diluent
of standard
concentration
Table 2.6.30.-2
endotoxin
Added endotoxin Number of
Solution Solution
(IV/roL) replicates
Solution A = Solution of the preparation being examined at A Test solution/f None 4
the dilution, here designated 1, at which the test for
interfering factors was carried out, i.e. the highest B Test solution/ f, None 4
concentration (lowest dilution) for which the endoroxin e Test solution/ f, None 4
recovery is within 50-200 per cent. Standard
Solution B = 2-fold dilution of solution A, not exceeding the endotoxin at
D Test solution/ f 4
2 x LOD for the
MVD. test system
Solution e = 2-fold dilution of solution B, not exceeding the Standard
MVD. endotoxin at
E Test solution/ f¡ 4
2 x LOD for the
Solution D = solution A spiked with standard endotoxin: the test system
middle dose from endoroxin standard curve (R3). Standard
endotoxin at
Solution Ro = negarive control. F Test solution/f,
2 x LOD for the
4
Solutions RI -~ = solutions of standard endoroxin at the test system

concentrations used in the test for interfering facrors . Pyrogen·free saline None (negative
Ro 4
or test diluent control)
7-1 -2 Caleulatian and interpretatian Standard
AlI data ro be inc1uded in the data analysis are ro relate to Pyrogen-free saline endotoxin at
4
R, 0.5 x LOD for
cells for which the 2 criteria for the standard curve are or test diluent
the test system
satisfied. T he endoroxin equivalents recovery calculated from
Standard
the endotoxin equivalents concentration found in solution D Pyrogen-free saline endotoxin at
R, 4
after subtracting the endotoxin equivalents concentration or test diluent 1 x LOD for the
test system
found in solution A, is within the range of 50-200 per cent.
For each different cell source, e.g. individual donation, donor Standard
Pyrogen·free saline endotoxin at
pool, or cellline, use the endotoxin standard curve RI-~ to R, 4
or test diluent 2 x LOD for the
calculate the concentration of endoroxin equivalents in each test system
of the replicates of solutions A, B and C. The preparation Standard
Pyrogen·free saline endotoxin at
being examined complies with the requirements of the test R. or test diluent 4 x LOD for the
4
for a given cell source if the mean concentrations of test system
endoroxin equivalents measured in the replica tes of solutions
A, B and e, after correction for dilution and concentration,
Solution A = solution of the preparation being examined at
are all les s than the eLe specified for the preparation being
the dilution, here designated 1, at which the test for
examined. interfering facrors was completed.
7-1-3 Passlfail eriteria aJ the preparatian Solution B = solution of the preparation being examined at a
When cells from individual donors are used, the preparation dilution, here designated JI' not exceeding the MVD, chosen
being examined is required to comply with the test with the after a review of data from the product-specific validation,
cells from each of 4 different donors. If the preparation being e.g. 1:2 x MVD (i.e. a 2-fold dilution aboye the MVD).
examined passes the test with cells from 3 of the 4 donors
V -A400 Appendix XIV H 2014
Solution e = solution of the preparation being examined at a Table 2.6.30.-3

dilution, here designated 12, not exceeding the MVD, chosen Solution/dilution
Solution Number of replicates
after a review of data from the product-specific validation, factor
e.g. MVD. A
Solution of reference
4
lotlf.
Solution D = solution A spiked with standard endo1Oxin at 2
Solution of reference
x LOD for the test system (as determined in prepara10ry B
lotlr,
4
testing) . Solution of reference
e 4
Solution E = solution B spiked with standard endo1Oxin at 2 lotlr.
x LOD for the test system. o Solution of test
4
preparation/ f.
Solution F = solution e spiked with standard endo1Oxin at 2
Solution of test
x LOD for the test system. E 4
preparationF
Solution Ro = negative control. F
Solution of test
4
preparation/ r.
Solution R¡ = standard endo1Oxin at 0.5 x LOD for the test
Positive control
system. G
(standard endotoxin)
4
Solution R 2 = standard endo1Oxin at 1 x LOD for the test Diluent (negative
Ro 4
system. control)
Solution R3 = standard endotoxin at 2 x LOD for the test

system. Solutions A, B and e are solutions of the reference lot
Solution R¡ = standard endo1Oxin at 4 x LOD for the test diluted by the dilution fac1Ors, 11, 12 and!J, determined in the
system. test for interfering factors.
Solutions D, E and F are solutions of the preparation being
7-2-2 Caleulation and interpretation
examined diluted by the dilution factors,l¡, fz and 13,
All data to be included in the data analysis are to relate to
determined for the reference lot in the test for interfering
cells for which mean responses to solutions Ro-R¡ increase
fac1Ors.
progressively. The mean response to Ro may be equal to the
mean response 10 R¡. For each different cell source, Solution G is the positive test control for the viability of the
e.g. individual donation, donor pool, or cellline, the mean cells and is a standard endo1Oxin concentration that gives a
response 10 solution R 2 is 10 be greater than a positive cut-off clear positive response.
value. Data below this cut-off value are considered negative. Solution Ro is the diluent used to dilute the preparation
If the mean response to Rl or R2 exceeds the cut-off value, _ being examined and serves as the test blank.
the response to the solution chosen for the pass/fail decision 7-3-2 Caleulation and interpretation
must be negative (= pass). For each negative solution of the Ail data 10 be included in the data analysis are to relate 10
test preparation (A, B and e), the mean response 10 the cells for which solution G and at least one of solutions A, B
corresponding spiked solution (D, E or F respectively) is and e give a response that is greater than the basal release of
compared with the mean response to R3 to determine the the read-out (Solution Ro). For each different ceil source,
percentage spike recovery. The contaminant concentration of e.g. individual donation, donor pool, or ceilline, use the
the preparation being examined is less than the eLe for a standard curve for the read-out (a calibration curve in
given cell source if the solution of the test preparation duplicate with a blank and at least 4 geometricaily diluted
designated for the pass/fail-decision and the dilutions below concentrations of the standard for the chosen read-out) and
ail give negative results and the endo1Oxin spike recovery is calculate the mean responses of the replicates of solutions
within the range of 50-200 per cent. A-F. Sum the mean responses to solutions A, B and e and
7-2-3 Pass/fail eriteria 01 the preparation sum the mean responses to solutions D, E and F. Divide the
The criteria are the same as for method'A (se e 7-1-3). sum of the mean responses 10 solutions D, E and F by the
sum of the mean responses 10 solutions A, B and C.
7-3 Method C: reference lot comparison test
The preparation being examined complies with the test for a
Method e involves a comparison of the preparation being given cell source if the resulting value complies with a
examined with a validated reference lot of that preparation. defined acceptance criterion not exceeding 2.5.
The reference lot is selected according 10 criteria that have
been justified and authorised. The test is intended to be 7-3-3 Pass/fail eritena 01 the preparation
performed in cases where a preparation being examined The criteria are the same as for method A (see 7-1-3).
shows marked interference but cannot be diluted within the To quantifY more closely the level of contamination,
Mvp to overcome the interference because it contains or is Methods A, B and e may be performed using other dilutions
believed 10 contain non-endo1Oxin contaminants. Responses of the solution of the preparation being examined not
to non-endo1Oxin contaminants may dilute out more rapidly exceeding the MVD.
than responses to endotoxin, which makes it necessary to The 10110wing seetion is published lar information only.
perform the test at a range of dilutions that include minimum
dilution. The test procedure is described below and includes GUIDANCE NOTES
an example for the comparison of a test lot with the reference
lot. 1 Introduction
7-3-1 Test proeedure The monocyte activation test (MAT) is primarily intended 10
Using the validated test method, prepare the solutions shown be used as an alternative method 10 the rabbit pyrogen test.
in Table 2.6.30.-3 and culture 4 replica tes of each solution The MAT detects pyrogenic and pro-infiamma1Ory
with cells from each of 4 individual donors or a single pool contaminants, including endotoxins from gram-negative
or with ceils from 1 passage of a human monocytic ceil lineo bacteria and 'non-endotoxin' contaminants, including
pathogen-associated molecular patterns (PAMPs), derived
from gram-positive and gram-negative bacteria, viruses and
2014 Appendix XIV H V-A40 1
fungi, and product-related and process-related biological or K

chemical entities. M
Since non-endotoxin contaminants are a physico-chemically
diverse c1ass of molecules, and usually the nature of the K threshold pyrogenic dose of endotoxin per kilogram of
contaminant in a preparation being examined is unknown, body mas s;
M maximum recommended bolus dose of product per
the level of contamination is expressed either in endotoxin-
equivalent units, derived by comparison with responses to kilogram of body mass.
standard endotoxin, or by comparison with a reference lot of When the product is to be injected at frequent intervals or
the test preparation. infused continuously, M is the maximum total dose
In the MAT, responses to standard endotoxin usually dilute administered in a single hour periodo
out over approximatively 1 log and responses to products The endotoxin limits depends on the product and its route of
contaminated with non-endotoxin contaminants (alone or in administration and is stated in monographs .
combination with endotoxins) often show very steep dose- Values for K are suggested in Table 2.6.30.-4.
response curves, usually over only 1 or 2 dilution steps when
tested for their capability to stimulate monocytes. Frequently, Table 2.6.30.-4
the largest response to such contaminated products is
Route of administration K (IU of endotoxin per kilogram
obtained with undiluted solutions of preparations being of body mass)
examined or small dilutions of the preparations being Intravenous 5.0
examined. For this reason test solutions of preparations being Intravenous, for radiopharmaceuticals 2.5
examined that contain or may corttain non-endotoxin
contaminants have to be tested at a range of dilutions that Intrathecal 0.2
inc1udes minimum dilution.
2 Methods For other routes, the acceptance criterion for bacterial

endotoxins is generally determined on the basis of results
2-1 Information regarding the choice of methods obtained during the development of the preparation.
Methods A, B and e, are not normally applied where a test
preparation has the intrinsic activity of stimulating the release 2-3 lnformation regarding cryo-protectants
of the chosen read-out or where the test preparation is The infiuence of cryo-protectants, e.g. dimethyl sulfoxide
contaminated with the chosen read-out. In both cases, this (DMSO), and their residues in thawed cells, is to be
fact is to be addressed by modifying and validating the considered: DMSO is toxic to cells in culture and, even
chosen method accordingly. The product-specific validation when cells have been washed thoroughly, cryo-preservation
of the chosen method would be expected to identify the may have altered cell properties, e.g. cell membrane
frequency of non-responders to a particular permeability.
productlcontaminant(s) combination and to identify steps to 2-4 Interference testing
address this, e.g. screening of donors, increasing the number Where practicable, interference testing is performed on at
of donors per test, and setting pass/fail criteria of appropriate least 3 different lots of the preparation being examined.
stringency to maxirnise the likelihood of detecting Preparations being examined that show marked batch-to-
contaminated batches. Methods A and 13 are appropriate batch variation, that effectively renders each batch unique for
when responses to dilutions of a preparation being examined the purposes of interference testing, are to be subjected to
are broadly parallel to responses to dilutions of standard interference testing within each individual test,
endotoxin. Method B is a semi-quantitative test that can also i.e. concomitant validation.
be applied when responses to dilutions of a test preparation
Interference testing is preferably performed on batch es of the
are not parallel to responses to dilutions of standard -
preparation being examined that are free of endotoxins and
endotoxin.
other pyrogeniclpro-infiammatory contaminants and, where
Method e, the reference lot comparison test, was developed this is not practicable, none of the batches are to be heavily
to address extreme donor variability in responses to certain contaminated. If only 1 batch is available the validation has
productlcontaminant(s) combinations. In this regard, it to be performed on that batch in 3 independent tests.
should be noted that, while monocytes from most donors Precision parameters for reproducibility, e.g. ± 50 per cent,
respond in a broadly similar manner to bacterial endotoxin, are to be fulfilled .
responses of monocytes from different donors to non-
endotoxin contaminants can differ markedly, so that it is 3 Replacement of the rabbit pyrogen test by the
possible to identify non-responders to certain monocyte activation test
productlcontaminant(s) combinations.
As noted aboye, the monocyte activation test (MAT) is
2-2 Calculation Of Contaminant Limit Concentration primarily intended to be used as an altemative method to the
The acceptance criterion for a pass/fail decision is the rabbit pyrogen test. Monographs on pharmaceutical products
contaminant limit concentration (eLC), which is expressed intended for parenteral administration that may contain
in endotoxin equivalents per milligram or millilitre or in units pyrogenic contaminants require either a test for bacterial
of biological activity of the preparation being examined. endotoxins or a monocyte activation test.
Where an endotoxin limit concentration (ELC) has been As a general policy:
specified for a product, the eLe is the same as the ELe, - in any individual monograph, when a test is required, only
unless otherwise prescribed. The eLe is expressed in tem1S 1 test is inc1uded, either that for bacterial endotoxins or
of endotoxin equivalents. The eLe is calculated using the the MAT. Before inc1uding the MAT in a monograph,
following expression: evidence is required that 1 of the 3 methods described in
the MAT chapter can be applied satisfactorily to the
product in question;
V-A402 Appendix XIV J 2014
- the necessary information is sought from manufacturers. there is a linear relation between factor IIa activity and the
Companies are invited to provide any validation data that cleavage of the chromogenic substrate.
they have conceming the applicability of the MAT to the Reagents
substances and formulations of interest. Such data include
Viper venom specific factor 11 activator (Ecarin) A
details of sample preparation and of any procedures
protein derived from the venom of the saw-scaled viper
necessary to eliminate interfering factors. In addition, any
(Echis carinatus) which specifically activates factor II.
available parallel data for rabbit pyrogen testing that
Reconstitute according to the manufacturer's instructions .
would contribute to an assurance that the replacement of
Store the reconstituted preparation at 4 oC and use within
a rabbit pyrogen test by the MAT is appropriate, is to be
1 month.
provided.
Factor Ha chromogenic substrate Specific chromogenic
4 Validation of alternative methods substrate for factor IIa such as: H-D-phenylalanyl-L-pipecolyl-
Replacement of a rabbit pyrogen test by a MAT, or L-arginine-4-nitroanilide dihydrochloride, 4-toluenesulfonyl-
replacement of a method for detecting pro- glycyl-prolyl-L-arginine-4-nitroanilide, H-D-cyclohexylglycyl-o:-
inflammatory/pyrogenic contaminants by another method, is aminobutyryl-L-arginine-4-nitroanilide, D-cyclohexylglycyl-L-
to be regarded as the use of an altemative method in the alanyl-L-arginine-4-nitroanilide diacetate. Reconstitute
replacement of a pharmacopoeial test, as described in the according to the manufacturer's instructions.
General Norices: Dilution buffer Solution containing 6.06 gIL of
'The test and assays described are the official methods upon tris(hydroxymethyl)aminomethane R, 17.53 gIL of sodium
which the standards of the European Pharmacopoeia are chloride R, 2.79 gIL of (ethylenedinitrilo) tetra-acetic acid R and
based. With the agreement of the competent authority, 1 gIL of bovine albumin R or human albumin R. Adjust to
altemative methods of analysis may be used for control pH 8.4 if necessary, using hydrochloric acid R.
purposes, provided that the methods used enable an Method
unequivocal decision to be made as to whether compliance
Test solution Dilute the preparation to be examined with
with the standards of the monographs would be achieved if
dilution buffer to obtain a solution containing 0.015 IU of
the official methods were used. In the event of doubt or
factor II per millilitre. Prepare at least 3 further dilutions in
dispute, the methods of analysis of the European
dilution buffer.
Pharmacopoeia are alone authoritative.'
Reference solution Dilute the reference preparation to be
The following procedures are suggested for validating a
examined with dilution buffer to obtain a solution containing
method for the MAT other than the one indicated in the
0.015 IU of factor II per millilitre. Prepare at least 3 further
monograph:
dilutions in dilution buffer.
- the procedure and the materials and reagents used in the
Warm all solutions to 37 oC in a water-bath shortly before
method should be validated as described for the test
the test.
concemed;
The following working conditions apply to microtitre plates.
- the presence of interfering factors (and, if needed, the
If the assay is carried out in tu bes, the volumes are adjusted
procedure for removing them) should be tested on
while maintaining the proportions in the mixture .
samples of at least 3 production batches. '
Using a microtitre plate maintained at 37 oC, add 25 f.lL of
MAT should be applied to all new products intended for
each dilution of the test solution or the reference solution to
parenteral administration that have to be tested for the
each of a series of wells. To each well add 125 f.lL of dilution
presence of monocyte-activating contaminants according to
buffer, then 25 ~lL of ecarin and incubate for exactly 2 mino
the requirements of the European Pharmacopoeia.
To each well add 25 flL of factor na chromogenic substrate.
Read the rate of change of absorbance (2.2.25) at 405 nm
continuously over a period of 3 min and obtain the mean
J. Blood and Related Products rate of change of absorbance (M/min) . If continuous
Coagulants monitoring is not possib1e, read the absorbance at 405 nm at
suitable consecutive intervals, for instance 40 s, plot the
Al. Assay of human coagulation factor 11
absorbances against time on a linear graph and calculate
(Ph. Eur. method 2.7.18) M /min as the slope of the lineo From the M /min values of
Human coagulation factor II is assayed following specific each individual dilution of standard and test preparations,
activation to form factor IIa. Factor na is estimated by calculate the potency of the preparation to be examined and
comparing its activity 'in cleaving a specific chromogenic check the validity of the assay by the usual statistical methods
peptide substrate with the same activity of the Intemational (5.3).
Standard or of a reference preparation calibrated in
Intemational Units. A2. Assay of human coagulation factor VII
The Intemational Unit is the factor II activity of a stated (Ph Eur. method 2.7. 10)
amount of the Intemational Standard which consists of a Human coagulation factor VII is assayed by its biological
freeze-dried concentrate of human blood coagulation factor activity as a factor VIIa-tissue factor complex in the
n . The equivalen ce in Intemational Units of the activation of factor X in the presence of calcium ions and
Intemational Standard is stated by the World Health phospholipids. The potency of a factor VII preparation is
Organisation. estimated by comparing the quantity necessary to achieve a
The chromogenic assay method consists of 2 steps: snake certain rate of factor Xa formation in a test mixture
venom-dependent activation of factor II, followed by containing the substances that take part in the activation of
enzymatic cleavage of a chromogenic factor lIa substrate to factor X, and the quantity of the Intemational Standard, or
form a chromophore that can be quantified of a reference preparation calibrated in Intemational Units,
spectrophotometrically. Under appropriate assay conditions, required to produce the same rate of factor Xa formation.
2014 Appendix XIV J V-A403
The Intemational Unit is the factor VII activity of a stated 0.2-2 mmoIILitre. The substrate may also contain
amount of the Intemational Standard, which consists of appropriate inhibitors to stop further factor Xa generation
freeze-dried plasma. The equivalence in Intemational Units (addition of edetate).
of the Intemational Standard is stated by the World Health Assay procedure
Organisation.
Reconstitute the entire contents of one ampoule of the
Human coagulation factor VII conce11trate BRP is calibrated in reference preparation and the preparation to be examined by
Intemational Units by comparison with the Intemational adding the appropriate quantity of water R; use within 1 h.
Standard. Add sufficient prediluent to the reconstituted preparations to
The chromogenic assay method consists of 2 consecutive produce solutions containing between 0.5 IU and 2.0 IU of
steps: the factor VII-dependent activation of factor X reagent factor VII per milIilitre.
mixture containing tissue factor, phospholipids and calcium Prepare further dilutions of reference and test preparations
ions, folIowed by enzymatic eleavage of a chromogenic factor using an isotonic non-chelating buffer containing 1 per cent
Xa substrate into a chromophore that can be quantified of bovine or human albumin, buffered preferably between
spectrophotometricalIy. Under appropriate assay conditions, pH 7.3 and 8.0. Prepare at least three separate, independent
there is a linear relation between the rate of factor Xa dilutions for each material, preferably in duplicate. Prepare
formation and the factor VII concentration. The assay is the dilutions such that the final factor VII concentration is
summarised in the folIowing scheme. below 0.005 IU/mL.
Step 1 Prepare a control solution that in eludes alI components
except factor VII.
tissue factor, Ca 2+ Prepare al! dilutions in plastic tubes and use wilhin 1 h.
a) factor VII ------------:J.~ factor Vlla
Step 1 Mix dilutions of the factor VII reference
preparation and the preparation to be examined with an
factor Vlla, 2+ appropriate volume of the prewarmed coagulation factor
_ _ _______ ~Ca_______ ~,.~fuctorXa
b) factor X
reagent or a combination of its separate constituents, and
tissue factor/phospholipid
incubate the mixture in plastic tubes or microplate welIs at
37 ce. The concentrations of the various components during
Step 2 the factor Xa generation must be as specified above under
the description of the reagents.
chromogenic substrate factor xa. peptide + chromophore AlIow the activation of factor X to proceed for a suitable
time, usualIy terminating the reaction before the factor Xa
concentration has reached its maximal level in order to
Both steps employ reagents that may be obtained
obtain a satisfactory linear dose-response relationship .
commercialIy from a variety of sources. AIthough the
The activation time is also chosen to achieve linear
composition of individual reagents may be subject to sorne production of factor Xa in time. Appropriate activation times
variation, their essential features are described in the
are usualIy between 2 min and 5 min, but deviations are
folIowing specification. permissible if acceptable linearity of the dose-response
Reagents relationship is thus obtained.
The coagulation factor reagent comprises purified proteins Step 2 Terminate the activation by the addition of a
derived from human or bovine sources. These inelude factor prewarmed reagent containing a chromogenic substrate .
X and thromboplastin tissue factor/phospholipid as factor VII QuantifY the rate of substrate eleavage, which must be linear
activator. These proteins are partly purified and do not with the concentration of factor Xa formed, by measuring
contain impurities that interfere with the activation of factor the absorbance change at an appropriate wavelength using a
VII or factor X. Factor X is present in amounts giving a final spectrophotometer, either monitoring the absorbance
concentration during the first step of the assay of continuously, thus alIowing the initial rate of substrate
10-350 nmol/litre, preferably 14-70 nmoI/litre. eleavage to be calculated, or terminating the hydrolysis
Thromboplastin from natural sources (bovine or rabbit brain) reaction after a suitable interval by lowering the pH by the
or synthetic preparations may be used as the tissue addition of a suitable reagent, such as acetic acid (500 giL
factor/phospholipid component. Thromboplastin suitable for C ZH 4 0 z) or a citrate solution (1 moIIL) at pH 3. Adjust the
use in prothrombin time determination is diluted 1:5 to 1:50 hydrolysis time to achieve a linear development of
in buffer such that the final concentration of Ca z+ is chromophore with time. Appropriate hydrolysis times are
15-25 mmoIILitre. The final factor Xa generation is usualIy between 3 min and 15 min, but deviations are
performed in a solution containing human or bovine albumin permissible if better Iinearity of the dose-response
at a concentration such that adsorption losses do not occur relationship is thus obtained.
and which is appropriately buffered at pH 7.3-8 .0. In the Check the validity of the assay and calculate the potency of
final incubation mixture, factor VII must be the only rate- the test preparation by the usual statistical methods (for
limiting component and each reagent component must lack example, 5.3).
the ability to generate factor Xa on its own.
The second step comprises the quantification of the formed A3. Assay of factor VIII fraction (human coagulation
factor Xa employing a chromogenic substrate that is specific factor VIII)
for factor Xa. GeneralIy this consists of a short peptide of (Ph. Eur. method 2.7.4)
between three and five amino acids, bound to a chromophore Human coagulation factor VIII is assayed by its biological
group. On eleavage of this group from the peptide substrate, activity as a cofactor in the activation of factor X by activated
its absorption maximum shifts to a wavelength alIowing its factor IX (factor IXa) in the presence of calcium ions and
spectrophotometric quantification. The substrate is usualIy phospholipid. The potency of a factor VIII preparation is
dissolved in water R and used at a final concentration of estimated by comparing the quantity necessary to achieve a
certain rate of factor Xa formation in a test mixture absence of factor VIII. In the final incubation mixture, factor
containing the substances that take part in the activation of VIII must be the only rate-Iimiting component.
factor X, and the quantity of the International Standard, or The 2nd step comprises the quantification of the formed
of a reference preparation calibrated in International Units, factor Xa, employing a chromogenic substrate that is specific
required to produce the same rate of factor Xa formation. for factor Xa. GeneralIy this consists of a derivatised short
The International Unit is the factor VIII activity of a stated peptide of between 3 and 5 amino acids, joined to a
amount of the International Standard, which consists of a chromophore group. On cIeavage of this group from the
freeze-dried human coagulation factor VIII concentrate. peptide substrate, its chromophoric properties shift to a
The equivalence in International Units of the International wavelength aIlowing its spectrophotometric quantification.
Standard is stated by the WorId Health Organisation. The substrate must also contain appropriate inhibitors to
Human coagulation factor VIII BRP is calibrated in stop further factor Xa generation, e.g. chelating agents, and
International Units by comparison with the International to suppress thrombin activity.
Standard. Assay prodecure
The chromogenic assay method consists of 2 consecutive Reconstitute the entire contents of 1 ampoule of the
steps: the factor VIII-dependent activation of factor X in a reference preparation and of the preparation to be examined;
coagulation-factor reagent composed of purified components, use immediately. Add sufficient prediluent to the
and the enzymatic cIeavage of a chromogenic factor Xa reconstituted preparations to produce solutions containing
substrate to yield a chromophore that can be quantified 0.5-2.0 IU/mL.
spectrophotometricalIy. Under appropriate assay conditions, The prediluent consists of haemophilia A plasma, or of an
there is a linear relation between the rate of factor Xa artificialIy prepared reagent that contains sufficient von
fonnation and the factor VIII concentration. The assay is WilIebrand factor and that gives results that do not differ
summarised by the folIowing scheme. significantly from those obtained employing haemophilia
plasma. The prediluted materials must be stable beyond the
Step 1
time required for the assay.
Prepare further dilutions of the reference and test
factor X (activated) factor VIII .. factor Xa
preparations using a non-chelating, appropriately buffered
factor IXa, phospholipid, Ca 2+ solution, for example, trisChydroxymethyl)aminomethane or
imidazole, containing 1 per cent of human or bovine
albumin. Prepare at least 2 dilution series of at least 3 further
Step 2 dilutions for each materia!. Prepare the dilutions such that
the final factor VIII concentration in the reaction mixture is
chromogenic substrate factor Xa.. peptide + chromophore preferably below 0.01 IU/mL, during the step offactor Xa
generation.
Prepare a control solution that incIudes alI components
Both steps employ reagents that may be obtained except factor VIII.
commercialIy from a variety of sources. Although the
Prepare aIl dilutions in plastic tubes and use immediately.
composition of individual reagents may be subject to sorne
variation, their essential features are described in the Step 1 Mix prewarmed dilutions of the factor VIII
folIowing specification. Deviations from this description may reference preparation and of the preparation to be exarnined
be pennissible provided that it has been shown, using the with an appropriate volume of the prewarmed coagulation
International Standard for human blood coagulation factor factor reagent or a combination of its separate constituents,
VIII concentrate as the standard, that the results obtained do and incubate the mixture in plastic tubes or microplate weIls
not differ significantly. at 37 oc. AIIow the activation of factor X to proceed for a
suitable time, terrninating the reaction Cstep 2) when the
It is important to demonstrate by validation the suitability of
factor Xa concentration has reached approximately
the kit used, notably by checking the time course of factor
50 per cent of the maximal (plateau) leve!. Appropriate
Xa generation in order to determine the time taken to reach
activation times are usuaIly between 2 min and 5 mino
50 per cent of the maximal factor Xa generation.
Step 2 Terminate the activation by addition of a
Reagents prewarmed reagent containing a chromogenic substrate.
The coagulation factor reagent comprises purified proteins QuantifY the rate of substrate cIeavage, which must be linear
derived from human or bovine sources. These incIude factor with the concentration of factor Xa formed, by measuring
X, factor IXa, and a factor VIII activator, usualIy thrombin. the absorbance change at an appropriate wavelength using a
These proteins are partly purified, preferably to at least spectrophotometer, either monitoring the absorbance
50 per cent, and do not contain impurities that interfere with continuously, thus alIowing the inirial rate of substrate
the activation of factor VIII or factor X. Thrombin may be cIeavage to be calculated, or terminating the hydrolysis
present in its precursor fonn prothrombin, provided that its reaction after a suitable interval by lowering the pH by
activation in the reagent is sufficiently rapid to give almost addition of a suitable reagent, such as a 50 per cent V/V
instantaneous activation of factor VIII in the assay. solution of acetic acid, or a 1 M pH 3 citrate buffer solution.
Phospholipid may be obtained from natural sources or be Adjust the hydrolysis time to achieve a linear development of
syntheticalIy prepared, and must, to a substantial extent, chromophore over time. Appropriate hydrolysis times are
consist of the species phosphatidylserine. The components of usualIy between 3 min and 15 min, but deviations are
the complete reagent are usuaIly divided into at least 2 permissible if better Iinearity of the dose-response
separate reagents, each lacking the ability to generate factor relationship is thus obtained.
Xa on its own. One of the reagents contains calcium ions. Calculate the potency of the test preparation by the usual
After reconstitution, the reagents may be combined provided statistical methods Cfor example, 5.3).
that no substantial amounts of factor Xa are generated in the
A4. Assay of factor IX fraction (human coagulation enzymatic cleavage of a chromogenic factor Xa substrate to
factor IX) form a chromophore that can be quantified
(Ph. Eur. method 2. 7.11) spectrophotometrically. Under appropriate assay conditions,
The principIe of the assay is to measure the ability of a factor there is a linear relation between factor Xa activity and the
IX preparation to reduce the prolonged coagulation time of cleavage of the chromogenic substrate.
factor IX-deficient plasma. The reaction is accelerated by Reagents
addition of a reagent containing phospholipid and a contact Russell's viper venom specific factor X activator (RVV)
activator, e.g. kaolin, silica or ellagic acid. The potency is A protein derived from the venom of Russell's viper (Vipera
assessed by comparing the dose-response curve of the russellz) which specifically activa tes factor X. Reconstitute
preparation to be examined to that of a reference according to the manufacturer's instructions. Store the
preparation, calibrated in International Units . reconstituted preparation at 4 oC and use within 1 month.
The International Unit is the factor IX activity of a stated Factor Xa chromogenic substrate Specific chromogenic
amount of the International Standard, which consists of a substrate for factor Xa such as: N-ex-benzyloxycarbonyl-D-
freeze-dried concentrate of human coagulation factor IX. arginyl-L-glycyl-L-arginine-4-nitroanilide dihydrochloride,
The equivalence in International Units of the International N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-4-
Standard is stated by the World Health Organisation. nitroanilide hydrochloride, methanesulfonyl-D-Ieucyl-glycyl-L-
Human coagulation factor IX concentrate BRP is calibrated in arginine-4-nitroanilide, methoxycarbonyl-D-cyclohexylalanyl-
International Units by comparison with the International glycyl-L-arginine-4-nitroanilide acetate. Reconstitute
Standard. according to the manufacturer's instructions.
Reconstitute separately the preparation to be examined and Dilution buffer Solution containing 3.7 gIL of
the reference preparation as stated on the label and use tris (hydroxymethyl) aminomethane R, 18.O giL of sodium
immediately. Where applicable, determine the amount of chloride R, 2.1 giL of imidazole R, 0.02 gIL of hexadimethrine
heparin present (2.7.12) and neutralise the heparin, for bromide R and 1 gIL of bovine albumin R or human
example by addition of protamine sulfate R (10 Ilg of albumin R. Adjust to pH 8.4 if necessary using hydrochloric
protamine sulfate neutralises 1 IU of heparin). Predilute the acid R.
preparation to be examined and the reference preparation in
Method
factor IX-deficient plasma (for example plasma substrate R2)
Test solution Dilute the preparation to be examined with
to produce solutions containing 0.5-2.0 IU/mL. Prepare at
dilution buffer to obtain a solution containing 0.18 IU of
least 3 dilutions for each 'material, preferably in duplicate,
using a suitable buffer solution (for examp1e imidazole buffer factor X per millilitre. Prepare at least 3 further dilutions in
solution pH 7.3 R) containing 10 gIL of bovine or human
dilution buffer.
albumin. Use these dilutions immediately. Reference solution Dilute the reference preparation to be
examined with dilution buffer to obtain a solution containing
Use an apparatus suitable for measurement of coagulation
times or carry out the assay with incubation tubes maintained 0.18 IU offactor X per millilitre. Prepare at least 3 further
in a water-bath at 37 oc. Place in each tube 0.1 mL of factor dilutions in dilution buffer.
IX-deficient plasma (for example plasma substrate R2) and Warm all solutions to 37 oC in a water-bath shortly before
0.1 mL of one of the dilutions of the reference preparation or the test.
of the preparation to be examined. Add to each tube 0.1 mL The following working conditions apply to microtitre plates.
of a suitable Activated Partial Thromboplastin Time (APTT) If the assay is carried out in tubes, the volumes are adjusted
reagent containing phospholipid and contact activator and while maintaining the proportions in the mixture.
incubate the mixture for a recommended time at 37 oC. Using a microtitre plate maintained at 37 oC, add 12.5 IlL of
To ea eh tube, add 0.1 mL of a 3.7 gIL solution of calcium each dilution of the test solution or the reference solution to
chloride R previously heated to 37 oc. Using a timer, measure each of a series of wells. To each well add 25 ~IL of RVV
the coagulation time, i.e. the interval between the moment of and incubate for exactly 90 S. To each well add 150 IlL of
the addition of the calcium chloride and the first indication factor Xa chromogenic substrate, diluted 1 in 6 in dilution
of the formation of fibrin. The volumes given aboye may be buffer.
adapted to the APTT reagent and apparatus used. Calculate Read the rate of change of absorbance (2.2.25) (at 405 nm
the potency using the usual statistical methods (for example,
continuously over a period of 3 min and obtain the mean
5.3). rate of change of absorbance (M/min). If continuous
monitoring is not possible, read the absorbance at 405 nm at
AS. Assay ofhuman coagulation factor X
suitable consecutive intervals, for instance 40 s, plot the
(Ph. Eur. method 2.7.19) absorbances against time on a linear graph and calculate
Human coagulation factor X is assayed following specific M/min as the slope of the lineo From the M /min values of
activation to form factor Xa. Factor Xa is estimated by ea eh individual dilution of standard and test preparations,
comparing its activity in cleaving a specific chromogenic calculate the potency of the preparation to be examined and
peptide substrate with the same activity of the International check the validity of the assay by the usual statistical methods
Standard or of a reference preparation calibrated in (5.3).
International Units.
The International Unit is the factor X activity of a stated A6. Assay of human coagulation factor XI
amount of the International Standard which consists of a (Ph. Eur. method 2.7.22)
freeze-dried concentrate of human coagulation factor X. The principIe of the assay is to measure the ability of a factor
The equivalence in International Units of the International XI preparation to reduce the prolonged coagulation time of
Standard is stated by the World Health Organisation. factor XI-deficient plasma. The reaction is accelerated by
The chromogenic assay method consists of 2 steps: snake addition of a reagent containing phospholipid and a contact
venom-dependent activation of factor X, followed by activator, e.g. kaolin, silica or ellagic acid. The potency is
assessed by comparing the dose-response curve of the by comparing, in given conditions, its activiry with the same
preparation ro be examined ro that of a reference preparation activiry of a reference preparation calibrated against the
consisting of human normal plasma. Inremational Standard, in Inremational Units where
1 unir of factor XI is equal to the activiry of 1 mL of human applicable.
normal plasma. Human normal plasma is prepared by The Intemational Unit is the activiry of a srated amount of
pooling plasma units from not fewer than 30 donors and the Inremational Standard, which consists of a freeze-dried
srored at - 30 oC or lower. human von Willebrand facror concenrrate. The equivalence
Reconstirute separately the preparation ro be examined and in Inremational Units of the Inremational Standard is stated
the reference preparation as stated on the label and use by the World Health Organisation (WHO).
immediately. Where applicable, determine the amount of Ristocetin cofactor assay
heparin presenr (2.7.12) and neutralise the heparin, for The ristocetin cofacror activiry of von Willebrand facror is
example by addition of protamine sulfate R (1 O ~lg of determined by measuring agglutination of a suspension of
proramine sulfate neutralises 1 IU of heparin). Predilute the platelets in the presence of risrocetin A. The assay can be
preparation to be examined and the reference preparation in carried out for quantitative determinations by using
facror XI-deficienr plasma (for example plasma substrate R3) automated instruments, or for semi-quanritative
ro produce solutions containing 0.5-2.0 units/rnL. Prepare at determinations by visually assessing the endpoinr of
least 3 appropriate dilutions for each material, preferably in agglutination in a dilution series. Quanritative assays are
duplica te, using a suitable buffer solution (for example preferred.
imidazole buffer solution pH 7.3 R) conraining 10 gIL of bovine
or human albumino Use these dilutions immediately. Reagents
Use an apparatus suitable for measurement of coagulation Suspension of platelets Use standardised and, for
times or perform the assay with incubation tubes maintained example, formaldehyde- or paraformaldehyde-fixed
in a water bath at 37 oc. Place in each tube 0.1 mL offacror preparations of freshly isolated and washed human platelets.
XI-deficienr plasma (for example plasma subsu"ate R3) and The suspension may also be freeze-dried. An appropriate
0.1 mL of one of the dilutions of the reference preparation or amount of ristocetin A is added if necessary. Sorne platelet
of the preparation to be examined. Add ro each tube 0.1 mL reagenrs may already conrain ristocetin A.
of a suitable Activated, Partial Thromboplastin Time (APTT) Reference preparation The reference preparation for von
reagenr conraining phospholipid and contact activator and Willebrand facror is the WHO Inremational Standard for
incubate the mixture for a recommended time at 37 oc. von Willebrand facror concentrate.
To each tube, add 0.1 mL of a 3.7 gIL solution of calcium Method
chloride R previously heated ro 37 oC . Using a timer, measure
Semi-quantitative assay Prepare suitable dilutions of the
the coagulation time, i.e. the inrerval between the momenr of
preparation to be examined and of the reference preparation,
the addition of the calcium chloride and the first indication
using as diluenr a solution conraining 9 gIL of sodium
of the formation of fibrin. The volumes given aboye may be
chlonde R and 10-50 gIL of human albumin R. Add ro each
adapted to the APTT reagenr and apparatus used. Calculate
dilution an appropriate amounr of the suspension of platelets
the potency using the usual statistical methods (for example,
and, if necessary, of risrocetin A. Mix on a glass slide by
5.3).
moving it gently in circles for 1 mino AlIow to stand for a
A 7. Activated coagulation factors further 1 min and read the result against a dark background
with side lighting. The last dilution which clearly shows
visible agglutination indicates the ristocetin cofactor titre of
Where applicable, determine the amounr of heparin present the sample. Use diluenr as a negative control.
(2.7.12) and neutralise the heparin, for example by addition
of protamine sulfate R (10 ¡.¡g of protamine sulfate neutra lis es Quantitative Assay Reconstitute the entire conrenrs of 1
1 IU of heparin). Prepare 1 to 10 and 1 ro 100 dilutions of ampoule of the reference preparation and the preparation to
the preparation to be examined using be examined by adding the appropriate quanrity of the
tris(hydroxymethyl)aminomethane buffer solution pH 7.5 R.
recommended diluenr (for example water R);
Place a series of polysryrene tubes in a water-bath at 37 DC use immediately. Add sufficienr prediluenr to the
and add to each tube 0.1 mL of platelet-poor plasma R and reconstituted preparations to produce solutions containing
0.1 mL of a suitable dilution of a phospholipid preparation to 0.5-2.0 IU/mL. The prediluenr consists of an isotonic non-
act as a platelet substitute. AlIow to stand for 60 s. Add ro chelating buffer conraining, for example, 1-5 per cenr of
each tube either 0.1 mL of 1 of the dilutions or 0.1 mL of human or bovine albumin, and
the buffer solution (conrrol tube). To each tube add tris (hydroxymethyl)aminomethane or imidazole, appropriately
immediately 0.1 mL of a 3.7 giL solution of calcium buffered.
chloride R previously heated ro 37 oC, and measure, within The test is performed in accordance with the manufacturer's
30 min of preparing the original dilution, the time that instructions with at least 2 dilution series with as many
elapses between addition of the calcium chloride solution and dilutions as are needed to obtain a rotal of at least 3 differenr
the formation of a clot. The test is not valid unless the concenrrations in the linear range of the assay.
coagulation time measured for the control tube is 200 s to Check the validiry of the assay and calculate the potency of
350 S. the test preparation using the usual statistical methods (for
example,5.3).
A8. Assay of human von Willebrand factor
Collagen-binding assay
Collagen-binding is determined by an enzyme-linked
The biological functions of human von Willebrand factor are
immunosorbent assay on collagen-coated microtitre plates.
numerous. At present, its risrocetin cofacror activiry and its
The method is based on the specific binding of von
collagen binding activiry can be utilised for assays.
WiIlebrand factor to colla gen fibrils and the subsequent
The potency of human von Willebrand factor is determined
binding of polyclonal anti-von Willebrand factor antibody film and incubate at 37 oC for 2 h. Empty the welIs of the
conjugated to an enzyme, which on addition of a plate by inverting and draining on a paper towel.
chromogenic substrate yields a product that can be Add 250 ¡.tL of washing buffer. Empty the wells of the plate
quantitated spectrophotometrically. Under appropriate by inverting and draining on a paper towel. Repeat this
conditions, there is a linear relationship between von operation 3 times.
Willebrand factor collagen-binding and absorbance. Prepare a suitable dilution of the conjugate (for example, a
Reagents dilution factor of 1 to 4000) with PBS containing 5 gIL of
Collagen Use native equine or human fibrils of collagen bovine serum albwnin R and add 100 ¡.tL to each well. Cover
type 1 or 111. For ease of handling, collagen solutions may be the plate with plastic film and incubate at 37 cC for 2 h.
used. Empty the wells of the plate by inverting and draining on a
paper towel. Add 250 ¡.tL of washing buffer. Empty the wells
Collagen diluent Dissolve 50 g of glucose R in water R,
of the plate by inverting and draining on a paper towel.
adjust to pH 2.7-2.9 with 1 M hydrochloric acid and dilute to
Repeat this operation 3 times.
Add 100 ¡.tL of substrate solution to each of the wells and
Phosphate-buffered saline (PBS) Dissolve 8.0 g of
incubate at room temperature for 20 min in the dark.
sodiwn chloride R, 1.05 g of disodium hydrogen phosphate
Add 100 ~lL of 1 M hydrochlorie acid to each of the wells.
dihydrate R, 0.2 g of sodium dihydrogen phosphate R and 0.2 g
of potassium chlonde R in water R. Adjust to pH 7.2 using Measure the absorbance at 492 nm. Use the absorbance
1 M sodium hydroxide or 1 M hydroehlorie acid and dilute to values to estima te the potency of the preparation to be
1000 mL with water R. examined using the usual statistical methods (5.3).
Washing buffer PBS containing 1 giL of polysorbate 20 R. The assay is invalid if the absorbances measured for the
negative control s are greater than 0.05 .
Blocking reagent PBS containing 1 gIL of polysorbate
20 R and 10 gIL of bovine serum albumin R. A9. Test for prekallikrein activator
Dilution buffer PBS containing 1 giL of polysorbate 20 R (Ph Eur. method 2. 6. 15)
and 50 gIL of bovine serum albumin R . Prekallikrein activator (PKA) activa tes prekallikrein to
Conjugate Rabbit anti-human von Willebrand factor kaIlikrein and may be assayed by its ability to cleave a
serum horseradish peroxidase conjugate. Use according to chromophore from a synthetic peptide substrate so that the
the manufacturer's instructions. rate of cleavage can be measured spectrophotometrically and
Substrate solution Immediately before use, dissolve a the concentration of PKA calculated by comparison with a
tablet of o-phenylenediamine dihydrochloride and a tablet of reference preparation calibrated in Intemational Units .
urea hydrogen peroxide in 20 mL of water R or use a The Intemational Unit is the activity of a stated amount of
suitable volume of hydrogen peroxide. Protect from light. the International Standard which consists of freeze-dried
Microtitre plates Flat-bonomed polystyrene plates with prekallikrein activator. The equivalence in Intemational Units
surface properties optimised for enzyme irnmunoassay and of the International Standard is stated by the World Health
high protein-binding capacity. Organisation.
Method Reagents
Test solutions Reconstitute the preparation to be Prekallikrein activator in albumin BRP is calibrated in
examined as stated on the label. Dilute with dilution buffer Intemational Units by comparison with the Intemational
to produce a solution containing approximately 1 IU of von Standard.
Willebrand factor. Prepare 2 series of at least 3 further Buffer A Dissolve 6.055 g of
dilutions using dilution buffer. tn's(hydroxymethyl)aminomethane R, 1.17 g of sodium
Reference solutions Reconstitute the reference ehloride R, 50 mg of hexadimethrine bromide R and 0.100 g of
preparation as directed. Dilute with dilution buffer to sodium azide R in water R . Adjust to pH 8.0 with 2 M
produce a solution containing approximately 1 IU of von hydrochloric acid R and dilute to 1000 mL with water R.
WilIebrand factor. Prepare 2 series of at least 3 further Buffer B Dissolve 6.055 g of
dilutions using dilution buffer. tris(hydroxymethyl)aminomethane R and 8.77 g of sodium
Allow the solution of collagen to warm to room temperature. ehloride R in water R. Adjust to pH 8.0 with 2 M hydroehloric
Dilute with collagen diluent to obtain a solution containing acid R and dilute to 1000 mL with water R.
30-75 ~lg/mL of collagen, mix gently to produce a uniform Preparation of prekallikrein substrate
suspension of collagen fibrils . Pipette 100 ¡.tL into each well
To avoid eoagulation activation, blood 01' plasma used for the
of the microtitre plate. Cover the plate with plastic film and
preparation of prekallikrein must come into contact only with
incubate at 37 oC ovemight. Empty the wells of the collagen-
plasties 01' silicone-treated glass suifaees.
coated plate by inverting and draining on a paper towel.
Add 250 ¡.tL of washing buffer. Empty the wells of the plate Draw 9 volumes of human blood into 1 volume of
by inverting and draining on a paper towel. Repeat this anticoagulant solution (ACD, CPD or 38 giL solution of
operation 3 times. Add 250 ¡.tL of blocking reagent to each sodium eitrate R) to which 1 mglmL of hexadimethrine
well, cover the plate with plastic film and incubate at 37 oC bromick R has been added. Centrifuge the mixture at 3600 g
for 1 h. Empty the wells of the plate by inverting and for 5 mino Separate the plasma and centrifuge again at
draining on a paper towel. Add 250 ~tL of washing buffer. 6000 g for 20 min to sediment plateIets . Separate the
Empty the wells of the plate by inverting and draining on a platelet-poor plasma and dialyse against 10 volumes of buffer
paper towel. Repeat this operation 3 times. A for 20 h. Apply the dialysed plasma to a chromatography
column containing agarose-DEAE for ion exehange
Add 100 ~lL each of the test solutions or reference solutions
chl'Omatography R which has been equilibrated in buffer A
to the welIs. Add 100 ¡.tL of dilution buffer to a series of
and is equal to twice the volume of the plasma. Elute from
wells to serve as negative control. Cover the plate with plastic
the column with buffer A at 20 mUcm 2 /h. Collect the eluate
in fractions and record the absorbance at 280 nm (2.2.25). methods are available. Procedures and reagents may vary
Pool the fractions containing the first protein peak so that the between different kits and the manufacturer's instructions are
volume of the pool is about 1.2 times the volume of the folJowed. The essential features of the procedure are
platelet-poor plasma. described in the folJowing example of a microtitre-plate
Test the substrate pool for absence of kalJikrein activity by kinetic method.
mixing 1 part with 20 parts of the pre-warmed chromogenic Reagents
substrate solution to be used in the assay and incubate at Dilution buffer pH 7.5 According to the manufacturer's
37 °C for 2 mino The substrate is suitable if the increase in instructions, a suitable buffer is used. Adjust the pH if
absorbance is less than 0.001 per minute. Add to the pooled necessary.
solution 7 gIL of sodium chloride R and filter through a
Plasmin A preparation of human plasmin that does not
membrane filter (nominal pore size 0.45 ¡.1m) . Freeze the
contain significant amounts of other proteases is preferably
filtrate in portions and store at - 25 oC; the substrate may
used. Reconstitute and store according to the manufacturer's
be freeze-dried before storage.
instructions.
Carry out alJ procedures from the beginning of the
Plasmin chromogenic substrate A suitable specific
chromatography to freezing in portions during a single
chromogenic substrate for plasmin is used: H-D-
working day.
cycJohexylalanyl-norvalyI-lysyl-p-nitroaniJine hydrochloride
Method (H-D-CHA-Nva-Lys-pNA.HCI) or L-pyroglutamyl-L-
The assay may be carried out using an automated enzyme phenylalanyl-L-lysyl-p-nitroaniJine hydrochloride (Glp-Phe-
analyser or a suitable microtitre pI ate system alJowing kinetic Lys-pNA.HCI) . Reconstitute in water R to give a suitable
measurements, with appropriate software for calculation of concentration according 10 the manufacturer's instructions.
results. Standards, samples and prekalJikrein substrate may
Method
be diluted as necessary using buffer B.
Varying quantities of the preparation to be examined are
Incubate diluted standards or samples with prekalJikrein mixed with a given quantity of plasmin and the remaining
substrate for 10 min such that the volume of the undiluted plasmin activity is determined using a suitable chromogenic
sample do es not exceed 1/10 of the total volume of the substrate.
incubation mixture to avoid errors caused by variation in
Reconstitute or thaw the preparation to be examined
ionic strength and pH in the incubation mixture. Incubate
according to the manufacturer's instructions. Dilute with
the mixture or a part thereof with at· least an equal volume of
dilution buffer pH 7.5 and prepare at least 2 independent
a solution of a suitable synthetic chromogenic substrate,
series of 3 or 4 dilutions for both the preparation 10 be
known to be specific for kalJikrein (for example, N-benzoyl-L-
examined and the reference standard.
prolyl-L-phenylalanyl-L-arginine 4-nitroanilide acetate R or D-
prolyl-L-phenylalanyl-L-arginine-4-nitroanilide- Mix 0.020 mL of each dilution with 0.020 mL of dilution
dihydro~hloride R), dissolved in buffer B. Record the rate of buffer pH 7.5 and warm 10 37 oC . Add 0.040 mL of a
change in absorbance per minute for 2-10 min at the plasmin solution (test concentration in the range of 0.2
wavelength specific for the substrate used. Prepare a blank nkatlrnL to 1.6 nkatlmL) previously heated 10 37 cC and
for each mixture of sample or standard using buffer B instead leave at 37 oC for 1 mino Add 0.020 mL of the chromogenic
of prekalJikrein substrate. substrate solution, previously heated to 37 oC, to each
mixture. Irnrnediately start measurement of the change in
Depending on the method used, M lmin has to be corrected
absorbance at 405 nrn (2.2.25) using a microtitre plate
by subtracting the value obtained for the corresponding blank
reader. Calculate the rate of change of absorbance (M/tnin).
without the prekalJikrein substrate. The results may be
Altematively, an end-point assay might be used by stopping
calculated using a standard curve, a parallel-line or a slope
ratio assay or any other suitable statistical method. Plot a the reaction with acetic acid and measuring the absorbance at
calibration curve using the values thus obtained for the 405 nm.
reference preparation and the respective concentrations; In both cases the duration of the cJeavage of the chromogenic
use the curve to determine the PKA activity of the substrate should be chosen to produce a linear increase in
preparation to be examined. absorbance at 405 nm, before substrate depletion becomes
significant. If the assay is performed in test tubes or cuvettes
AlO. Assay of huptan plasmin inhibitor using a spectrophotometric method, the volumes of reagent
(Ph. Eur. method 2.7.25) solutions are changed proportionalJy.
Human plasmin inhibitor, also calJed human cx2-antiplasmin, Substract the optical density of the blank (prepared with
is a plasma protein that inhibits the plasmin (a serine dilution buffer pH 7.5) from the optical density of the
protease) pathway of fibrinolysis by rapidly forming a preparation to be examined. Check the validity of the assay
complex with free plasmin. Furthermore, upon blood and calculate the potency of the preparation 10 be examined
coagulation, human plasmin inhibitor is cross-linked to fibrin by the usual statistical methods (5.3).
strands by factor XIII, and interferes with binding of the
Anticoagulants
proenzyme plasminogen to fibrin.
The potency of human plasmin inhibitor is estimated by Bl. Assay of heparin in coagulation factors
comparing the ability of the preparation to be examined to (Ph. Eur. method 2.7.12)
inhibit the cJeavage of a specific chromogenic substrate by Heparin is assayed as a complex with antithrombin III (AT)
plasmin with the same ability of a reference standard of via its inhibition of coagulation factor Xa (anti-Xa activity).
human plasmin inhibitor. Plasmin cJeavage of the An excess of AT is maintained in the reaction mixture 10
chromogenic substrate yields a chromophore that can be ensure a constant concentration of the heparin-AT complex.
quantified spectrophotometricalJy. Factor Xa is neutralised by the heparin-AT complex and the
The individual reagents for the assay may be obtained residual factor Xa hydrolyses a specific chromogenic peptide
separately or in commercial kits. Both end-point and kinetic substrate to release a chromophore. The quantity of
chromophore is inversely proportional to the activity of the Hepann sodium BRP is calibrated in Intemational Units by
heparin. comparison with the Intemational Standard by means of the
Factor Xa chromogenic substrate Specific chromogenic assay given below.
substrate for factor Xa such as: N-benzoyl-L-isoleucyl-L- Carry out the assay using one of the following methods for
glutamyl-glycyl-L-arginine-4-nitroanilide hydrochloride. determining the onset of c10tting and using tubes and other
Reconstitute according to the manufacturer's instructions. equipment appropriate to the chosen method:
Dilution buffer 6.05 gIL solution of a) direct visual inspection, preferably using indirect
tns(hydroxymethyl)aminomethane R . Adjust to pH 8.4 if illumination and viewing against a matt black
necessary using hydroehlone acid R. background;
Test solution Dilute the preparation to be examined with b) spectrophotomettic recording of the change in optical
dilution buffer to obtain a solution expected to contain density at a wavelength of approximately 600 nm;
0.1 IU of heparin per millilitte. c) visual detection of the change in fiuidity on manual
Reference solution Dilute the heparin reference tilting of the tubes;
preparation with dilution buffer to obtain a solution d) mechanical recording of the change in fiuidity on
containing 0.1 IU of heparin per millilitte. stirring, care being taken to cause the minimum
The following working conditions apply to microtitte plates. disturbance of the solution during the earliest phase of
If the assay is carried out in tubes, the volumes are adjusted c1otting.
while maintaining the proportions in the mixture . Assay procedure
Warm all solutions to 37 oC in a water-bath shortly before The volumes in the text are given as examples and may be
the test. adapted ro the apparatus used provided that the ratios between the
Disttibute in a series of wells, 20 ¡.¡L of normal human different volumes are respected.
plasma and 20 ¡.¡L of antithrombin JI! solution RI. Add to the Dilute hepann sodium BRP with a 9 giL solution of sodium
wells a series of volumes (20 ¡.tL, 60 ¡.¡L, 100 ¡.tL and 140 ¡.¡L) ehloride R to contain a precisely known number of
of the test solution or the reference solution and make up the Intemational Units per millilitre and prepare a similar
volume in each well to 200 ¡.tL using dilution buffer solution of the preparation to be examined which is expected
(0.02-0.08 IU of heparin per millilitte in the final reaction to have the same activity. Using a 9 gIL solution of sodium
mixture) . ehlonde R, prepare from each solution a series of dilutions in
End-point method Transfer 40 ¡.¡L from each well to a geometric progression such that the c10tting time obtained
second series of wells, add 20 ¡.tL of bovine ¡aeror Xa with the lowest coneentration is not less than 1.5 times the
solution R and incubate at 37 oC for 30 s. Add 40 ¡.tL of a blank reealcification time, and that obtained with the highest
1 mmollL solution of factor Xa chromogenic substrate and concentration is such as to give a satisfactory log dose-
incubate at 37 oC for 3 minoTerminate the reaction by response curve, as determined in a preliminary test.
lowering the pH by the addition of a suitable reagent, such Place 12 tubes in a bath of iced water, labelling them in
as a 20 per cent V/V solution of glacial aeetic aeid R and duplica te: T ¡, T 2 and T 3 for the dilutions of the preparation
measure the absorbance at 405 nm (2.2.25) . Appropriate to be examined and S¡, S2 and S3 for the dilutions of the
reaction times are usually between 3 min and 15 min, but reference preparation. To each tube add 1.0 mL of thawed
deviations are permissible if better linearity of the dose- plasma substrate RI and 1.0 mL of the appropriate dilution of
response relationship is thus obtained. the preparation to be examined or the reference preparation.
Kinetic method Transfer 40 ¡.tL from each well to a After each addition, mix but do not allow bubbles to formo
second series of wells, add 20 ~lL of bovine ¡aeror Xa Treating the tubes in the order S¡, S2' S3' T¡, T 2, T 3,
solution R and incubate at 37 oC for 30 s. Add 40 ~lL of a transfer each tube to a water-bath at 37 oC, allow to
2 mmollL solution of factor Xa chromogenic substrate, equilibrate at 37 oC for about 15 min and add to each tube
incubate at 37 oC and measure the rate of substrate cleavage 1 mL of a suitable APTT (Activated Partial Thromboplastin
by continuous measurement of the absorbance change at Time) reagent containing phospholipid and a contact
405 nm (2.2.25), thus allowing the initial rate of substrate activator, at a dilution giving a suitable blank recalcification
c1eavage to be calculated. This rate must be linear with the time not exceeding 60 s. After exactly 2 min add 1 mL of a
concentration of residual factor Xa. . 3.7 giL solution of ealcium ehlonde R previously heated to
Check the validity of the assay and calculate the heparin 37 oC and record as the clotting time the interval in seconds
activity of the test preparation by the usual statistical between this last addition and the onset of c10tting
methods for a slope-ratio assay (for example, 5.3) . determined by the ehosen technique . Determine the blank
recalcification time at the beginning and at the end of the
B2. Assay of heparin procedure in a similar manner, using 1 mL of a 9 giL
(Ph. Eur. method 2.7.5) solution of sodium ehlonde R in place of one of the heparin
The anticoagulant activity of heparin is determined in vitro by dilutions; the 2 blank values obtained should not differ
comparing its ability in given conditions to delay the c10tting significantly. Transform the c10tting times to logarithms,
of recalcified citrated sheep plasma with the same ability of a using the mean value for the duplicate tubes. Repeat the
reference preparation of heparin calibrated in Intemational procedure using fresh dilutions and carrying out the
Units. incubation in the order T¡, T z, T 3 , SI' S2' S3. Calculate the
results by the usual statistical methods (5.3).
The Intemational Unit is the activity contained in a stated
amount of the Intemational Standard, which consists of a Carry out not fewer than 3 independent assays. For ea eh
quantity of freeze-dried heparin sodium from pork intestinal such assay prepare fresh solutions of the referenee
mucosa. The equivalence in Intemational Units of the preparation and the preparation to be examined and use
Intemational Standard is stated by the World Health another, freshly thawed portion of plasma substrate.
Organisation.
V -A41 o Appendix XIV J 2014
Calculate the potency of the preparation to be examined, Step 1

combining the results of these assays, by the usual statistical
methods (5.3). When the variance due to differences between human protein C activator
assays is significant at P = 0.01, a combined estimate of human protein C ~ APC
potency may be obtained by calculating the non-weighted
mean of potency estimates.
Step 2
B3. Assay of human antithrombin III
(Ph. Eur. method 2.7.17) APC
chromogenic substrate _ _ _ peptl'd e + chromop hore
_--'.
The antithrombin III content of the preparation to be
examined is detennined by comparing its ability to inactivate The potency of human protein C is estimated by comparing
thrombin in the presence of an excess of heparin with the the ability of the preparation to be examined to cleave a
same ability of a reference preparation of human chromogenic substrate with the same ability of a reference
antithrombin II! concentrate calibrated in Intemational standard of human protein C calibrated in International
Units. Varying quantities of the preparation to be examined Units. The International Unit is the activity of a stated
are mixed with a given quantity of thrombin and the amount of the Intemational Standard for human protein C.
remaining thrombin activity is detennined using a suitable The equivalence in International Units of the Intemational
chromogenic substrate. Standard is stated by the World Health Organisation.
The Intemational Unit is the activity of a stated amount of Individual reagents may be obtained separately or in
the Intemational Standard for human antithrombin II! commercial kits. Both end-point and kinetic methods are
concentrate. The equivalence in Intemational Units of the available. Pro ce dures and reagents may vary between
Intemational Standard is stated by the World Health different kits and the manufacturer's instructions are
Organisation. followed. The essential features of the procedure are
Method described in the following example of a microtitre plate
Prepare 2 independent series of 3 or 4 dilutions in the range end-point method.
1/75 to 1/200 from 1 IU/mL, for both the preparation to be Reagents
examined and the reference preparation, using tris-EDTA Dilution buffer pH 8.4 Dissolve 6.055 g of
ESA buffer solution pH 8.4 R containing 15 IU of heparin per tris(hydroxymethyl)aminomethane R and 16.84 g of eaesium
millilitre. ehlonde R in water R and adjust the pH if necessary. Dilute
Warm 200 ¡.¡L of each dilution at 37 oC for 1-2 mino Add to to 1000.0 mL with water R.
each dilution 200 ¡.¡L of a solution of bovine thrombin R Human protein e activator Protein isolated from the
containing 2 IU/mL in tris-EDTA ESA buffer solution venom of the viper Agkistrodon eontortrix eontortrix that
pH 8.4 R. Mix and maintain at 37 oC for exactly 1 mino specifically activates human protein C. Reconstitute and
Add 500 ¡.¡L of a suitable chromogenic substrate (for store according to the manufacturer's instructions. Dilute to
example, D-phenylalanyl-L-pipecolyl-L-arginine-4-nitroanilide, 0.25 U/mL with water R before use in the assay.
reconstituted in water R to give a solution containing
Activated protein e chromogenic substrate Specific
4 mmol/L and further diluted to a concentration suitable for
chromogenic substrate for APC, for example L-pyroglutamyl-
the assay using tris-EDTA ESA buffer solution pH 8.4 R
L-prolyl-L-arginyl-p-nitroaniline hydrochloride (pyroGlu-Pro-
without albumin). Immediately start measurement of the
Arg-pNA.HCl) . Reconstitute with water R to give a
change in absorbance at 405 nm (2.2.25), continuing the
concentration of 4.5 mmollL. Further dilute to 1.1 mmollL
measurement for at least 30 S. Calculate the rate of change of
with dilution buffer pH 8.4 before use in the assay.
absorbance (M/min). (Altematively, an end-point assay may
be used by stopping the reaction with acetic acid and Method
measuring the absorbance at 405 nro.) Reconstitute or thaw the preparation to be examined
The rate of change of absorbance (M/min) is inversely according to the manufacturer's instructions. Dilute with
proportional to antithrombin III activity. water R to produce at least 3 separate dilutions for each
Check the validity of the assay and calculate the potency of preparation in the range 0.050-0.200 IU/roL, preferably in
the test preparation by the usual statistical methods (5.3). duplicate.
Step 1 Mix 0.025 mL of each dilution with 0.050 mL of
B4. Assay of human protein e the human protein C activator, both previously heated to
(Ph . Eur. method 2.7.30) 37 oC, and leave at 37 oC for exactly 10 mino For each
dilution, prepare a blank in the same manner, using water R
1. CHROMOGENIC ASSAY
instead of the human protein C activator.
Human protein C is a vitamin K-dependent plasma protein
that, upon activation to activated protein C (APC), can Step 2 Add 0.150 mL of diluted chromogenic substrate,
inhibit blood coagulation through the proteolytic cleavage of previously heated to 37 oC, to each mixture and leave at
factors Va and VIIIa. Human protein C activity is estimated 37 oC for exactly 10 mino The incubation time must be
using a two-step method: in the 1sr step, human protein C in adjusted, if necessary, to ensure a linear development of
the preparation is activated by a specific activator from snake chromophore with time. Terminate the reaction by adding
venomj in the 2nd step, APC cleaves a specific chromogenic 0.050 mL of a 50 per cent V/V solution of glacial aeetie
substrate to fonn a chromophore that can be quantified acid R.
spectrophotometrically. Cleavage of the chromogenic substrate by APC causes release
of the chromophore pNA, in proportion to the concentration
of human protein C in the preparation. Measure the optical
density at a wavelength of 405 nm. Subtract the optical
density of the blank from the optical density of the test
sample. eheck the validity of the assay and calculate the sensitive to the ability of human protein S to accelerate the
potency of the preparation to be examined using the usual inactivation of factor Va by APe. In practice, the assay
statistical methods (5.3). involves the addition of human protein S to a reagent
2. CLOTTING ASSA y mixture containing APe, factor Va and human protein
Human protein e activity is estimated following cleavage to S-deficient plasma. Prolongation of the clotting time is
APe by a specific activator extracted from the venom of the proportional to the concentration of human protein S in the
viper Agkistrodon eontortrix eontortrix. The resulting APe preparation. Methods in which APe is added directly as a
inactivates factors Va and VIlla, and thus prolongs the reagent are preferred to those in which APe is generated
APTT (Activated Partial Thromboplastin Time) of a system during the assay by the addition of a specific human protein
in which all the coagulation factors are present, constant and e activator purified from snake venom. Activation of
in excess, except for human protein e, which is derived from coagulation is initiated by the addition of an activating
the preparation being tested. Prolongation of the clotting reagent such as thromboplastin or activated factor X,
time is proportional to the concentration of human protein e together with phospholipids and calcium chloride. During the
in the preparation. assay, factor Va is generated from factor V in the human
protein S-deficient plasma following the activation of
The potency of human protein e is estimated by comparing
coagulation. The assay procedure must ensure that human
the ability of the preparation to be examined to pro long the
protein S is the only limiting factor.
clotting time with the same ability of a reference standard of
human protein e calibrated in International Units. The potency of human protein S is estimated by comparing
The International Unit is the activity of a stated amount of the ability of the preparation to be examined to pro long the
the International Standard for human protein e. clotting time with the same ability of a reference standard of
The equivalence in International Units of the International human protein S calibrated in International Units.
Standard is stated by the World Health Organisation. The International Unit is the activity of a stated amount of
the International Standard for human protein S.
Individual reagents may be obtained separately or in
The equivalence in International Units of the International
commercial kits. Procedures and reagents may vary between
Standard is stated by the World Health Organisation.
different kits and the manufacturer's instructions are
followed. The essential features of the procedure are Individual reagents may be obtained separately or in
described in the following example. commercial kits. Procedures and reagents may vary between
different kits and the manufacturer's instructions are
Reagents followed. The essential features of the procedure are
Dilution buffer pH 7.4 Isotonic non-chelating buffer. described in the following example.
Human protein C-deficient plasma eitrated human Reagents
plasma with no measurable human protein e content.
Dilution buffer pH 7.4 Isotonic non-chelating buffer
Reconstitute and store according to the manufacturer's
prepared as follows : dissolve 6.08 g of
instructions.
tris(hydroxymethyl)aminomethane R and 8.77 g of sodiwn
Human protein C activator .Protein isolated from the ehlonde R in water R and adjust the pH if necessary;
venom of the viper Agkistrodon contortrix contortrix that add 10 g of bovine albumin R or human albumin R and dilute
specifically activa tes human protein e. Reconstitute and to 1000.0 mL with water R.
store according to the manufacturer's instructions.
Human protein S-deficient plasma eitrated human
Coagulation activator A suitable APTT reagent plasma with no measurable human protein S content and,
containing phospholipids and a contact activator may be preferably, also free of e4b-binding protein.
used. It may be combined with the human protein e
Coagulation activator This reagent is used to initiate
activator.
coagulation in the human protein S-deficient plasma, and
Method thereby also provides a source of activated factor V.
Reconstitute or thaw the preparation to be examined The activator may consist of tissue factor, activated factor X,
according to the manufacturer's instructions . Dilute with or an agent capable of directly activating factor X that may
dilution buffer pH 7.4 to produce at least 3 separate dilutions be purified from the venom of Russell's viper (Vipera russellt).
for each preparation in the range 0.010-0.150 IU/mL, The reagent also contains APe, phospholipids and ealcium
preferably in duplicate. ehloride R, or, alternatively, calcium chloride may be added
Mix 1 volume of each dilution with 1 volume of human separately after a timed activation periodo
protein e-deficient plasma and 1 volume of the human Method
protein e activator (combined with the APTT reagent where Reconstitute or thaw the preparation to be examined
appropriate), all previously heated to 37 °e. Add 1 volume of according to the manufacturer's instructions. Dilute with
0.025 M ealcium ehloride solution R previously heated to dilution buffer pH 7.4 to produce at least 3 separate dilutions
37 °e, and record the clotting time. for each preparation in the range 0.020-0.100 IU/rnL,
The clotting time is proportional to the concentration of preferably in duplicate.
human protein e in each dilution. eheck the validity of the Mix 1 volume of each dilution with 1 volume of human
assay and calculate the potency of the preparation to be protein S-deficient plasma, both previously heated to 37 ce.
examined using the usual statistical methods (5.3) . Add 2 volumes of the coagulation activator, previously heated
to 37 °e, and record the clotting time.
B5. Assay of human protein S
Alternative pro ce dures may use a coagulation activator
without calcium chloride, and require a precisely timed
Human protein S is a vitamin K-dependent plasma protein
activation period before the addition of calcium chloride and
that acts as a cofactor for the anticoagulant functions of
the measurement of clotting time.
activated protein e (APC). Human protein S activity may be
determined by the clotting assay described below, which is
V -A412 Appendix XIV J 2014
The clotting time is proportional to the concentration of Antigen coating of tanned human red blood cells Take
human protein S in each dilution. Check the validity of the a suitable volume (Vs) of tanned cells, add 0.2 mL of rubella
assay and calculate the potency of the preparation to be antigen per 1.0 mL of tanned cells and incubate at 37 oC for
examined using the usual statistical methods (5.3). 30 mino Collect the cells by centrifugation (800 g for
10 min) and discard the supernatant. Add a volume of
Immunoglobulins albumin barbital buffer solution equivalent to the discarded
supernatant, resuspend and collect the cells as described and
el. Test for Fe funetion of immunoglobulin repeat the washing procedure. Resuspend with albumin
(Ph. Eur. method 2.7.9) barbital buffer solution using a volume equivalent to 3/4 of
The test lor Fe lunetion 01 immunoglobulin is earried out using V" thereby obtaining the initial volume (V¡) . Mix 900 ¡¡L of
method A or B. Method B is an adaptation 01 the proeedure 01 albumin barbital buffer solution with 100 ¡¡L of Vi> which is
method A lor the use 01 microtitre plates lor the measurement 01 thereby reduced to the residual volume (Vr), and determine
eomplement-mediated haemolysis. Differenees in the test proeedures the initial absorbance at 541 nm (A). Dilute V;. by a factor
between methods A and B are addressed in the test. equal to A using albumin barbital buffer solution, thereby
obtaining the final adjusted volume V¡ = Vr x A of
Reagents
sensitised human red blood cells and adjusting A to
Stabilised human blood Collect group O human blood 1.0 ± 0.1 for a tenfold di1ution.
into ACD anticoagulant solution. Store the stabilised blood
Antibody binding of antigen-coated tanned human red
at 4 oC for not more than 3 weeks.
b100d cells Prepare the following solutions in succession
Phosphate-buffered saline pH 7.2 Dissolve 1.022 g of and in duplica te, using for each solution a separate half-
anhydrous disodium hydrogen phosphate R, 0.336 g of micro cuvette (for example, disposable type) or test-tube.
anhydrous sodium dihydrogen phosphate R and 8.766 g of
(1) Test solutions If necessary, adjust the
sodium ehloride R in 800 mL of water R and dilute to
immunoglobulin to be examined to pH 7.
1000 rnL with the same solvento
Where method A is performed, dilute volumes of the
Magnesium and calcium stock solution Dissolve
preparation to be examined with albumin barbital buffer to
1.103 g of ealcium ehloride R and 5.083 gof magnesium
obtain 30 mg and 40 mg of immunoglobulin and adjust the
ehloride R in water R and dilute to 25 mL with the same
volume to 900 ¡¡L with albumin barbital buffer.
solvento
Where method B is performed, dilute volumes of the
Barbital buffer stock solution Dissolve 207 .5 g of sodium
preparation to be examined with albumin barbital buffer to
ehloride R and 25.48 g of barbital sodium R in 4000 mL of
obtain 15 mg and 30 mg of immunoglobulin and adjust the
water R and adjust to pH 7.3 using 1 M hydroehlorie aeid.
volume to 1200 ~IL with albumin barbital buffer.
Add 12.5 mL of magnesium and calcium stock solution and
dilute to 5000 mL with water R. Store at 4 oC in transparerit (2) Reference solutions Prepare as for the test solutions
containers. using human immunoglobulin (Fe funetion and molecular
size) BRP.
Albumin barbital buffer solution Dissolve 0.150 g of
bovine albumin R in 20 mL of barbital buffer stock solution (3) Complement control Albumin barbital buffer
and dilute to 100 mL with water R . Prepare immediately solution.
before use. Where method A is performed, add to each cuvetteltest-tube
Tannic acid solution Dissolve 10 mg of tannie acid R in 100 ¡¡L of sensitised human red blood cells and mix well.
100 mL of phosphate-buffered saline pH 7.2. Prepare Allow to stand for 15 min, add 1000 ¡.tL of albumin barbital
immediately before use. buffer solution, collect the cells by centrifugation (1000 g for
10 min) of the cuvette/test-tube and remove 1900 ¡¡L of the
Guinea-pig complement Prepare a pool of serum from
supernatant. Replace the 1900 ~IL with albumin barbital
the blood of not fewer than 10 guinea-pigs. Separate the
buffer solution and repeat the whole of the washing
serum from the clotted blood by centrifugation at about
procedure, finally leaving a volume of 200 ¡lL. T est samples
4 oC. Store the serum in small amounts below -70 oC.
may be stored in sealed cuvettes/test-tubes at 4 oC for not
Irnmediately before starting complement-initiated haemolysis,
longer than 24 h.
dilute to 125-200 CH so per millilitre with albumin barbital
buffer solution and store in an ice-bath during the test. Where method B is performed, add to each test-tube 300 ¡¡L
of sensitised human red blood cells and mix well (the final
Rubella antigen Suitable rubella antigen for
irnmunoglobulin concentration is in the range of
haemagglutination-inhibition titre (HIT). Titre > 256 HA
10-20 mg/mL) . Allow to stand for 15 min, add 1500 ¡¡L of
units.
albumin barbital buffer solution and stir gently until
Preparation oftanned human red blood cells Separate homogeneous. Collect the cells by centrifugation (1000 g for
human red blood cells by centrifuging an appropriate volume 10 min) of the test-tube, remove the supernatant and add
of stabilised human blood, wash the cells at least 3 times approximately 3 mL of albumin barbital buffer solution.
with phosphate-buffered saline pH 7.2 and suspend at Repeat this operation up to 4 times in total, leaving a final
2 per cent VIV in phosphate-buffered saline pH 7.2. volume of 300 ¡¡L. Test samples may be stored in sealed test-
Add 0.2 mL oftannic acid solution to 14.8 mL of tubes at 4 oC for not longer than 24 h.
phosphate-buffered saline pH 7.2. Mix 1 volume of the
Comp1ement-initiated haemo1ysis.
freshly prepared dilution with 1 volume of the human red
blood cell suspension and incubate at 37 oC for 10 mino To measure haemolysis where method A is performed, add
Collect the cells by centrifugation (800 g for 10 min), 600 ¡.tL of albumin barbital buffer solution warmed to 37 oC
discard the supernatant and wash the cells once with to the test sample, resuspend the cells carefully by repeated
phosphate-buffered saline pH 7.2. Resuspend the tanned pipetting (not fewer than 5 times) and place the cuvette in
cells at 1 per cent VIV in phosphate-buffered saline pH 7.2. the thermostatted cuvette holder of a spectrophotometer.
After 2 min, add 200 ¡¡L of diluted guinea-pig complement
(125-200 CH 50 /mL), mix thoroughly by pipetting twice and Magnesium and calcium stock solution Dissolve
start irnmediately after the second pipetting the time- 1.103 g of ealcium ehloride R and 5.083 g of magnesium
dependent recording of absorbance at 541 nm, using ehloride R in water R and dilure to 25 mL with the same
albumin barbital buffer solution as the compensation liquid. solvent.
Stop the measurement if absorbance as a function of time Barbital buffer stock solution Dissolve 207.5 g of sodium
has clearly passed the infiexion point. ehlaride R and 25.48 g of barbital sodium R in 4000 mL of
To measure haemolysis where method B is performed, add water R and adjust to pH 7.3 using 1 M hydroehlorie acid.
900 ¡.¡L of albumin barbital buffer solution warmed to 37 cC Add 12.5 mL of magnesium and calcium stock solution and
to each test-tube and resuspend the cells carefully by dilute to 5000 mL with water R. Filter through a membrane
repeated pipetting (not fewer than 5 times) . The microtitre filter (nominal pore size 0.22 ¡.¡m) . Store at 4 cC in glass
plate must be prewarmed to 37 oC before starting the test. containers.
Transfer 240 ~lL of each solution into 4 microtitre plate wells Gelatin solution Dissolve 12.5 g of gelatin R in about
then incubate the microplate at 37 oC for 6 min, stirring 800 mL of water R and heat to boiling in a water-bath. Cool
gently every lOs. To each microtitre plate well add 60 ¡.¡L of to 20 cC and dilute to 10 L with water R. Filter through a
diluted guinea-pig complement (150 CHso/mL). Mix for 10 s membrane filter (nominal pore size 0.22 ¡.¡m). Store at 4 oc.
and immediately start recording the absorbance at 541 nm at Use clear solutions only.
37 oC, measuring every 20 S. Stop the measurement if the Citrate solution Dissolve 8.0 g of sodium citrate R, 4.2 g
absorbance as a function of time has clearly passed the of sodium ehloride R and 20.5 g of glueose R in 750 mL of
infiexion point.
water R. Adjust to pH 6.1 using a 100 gIL solution of citrie
Evaluation For each cuvetteltest-tube/well, determine the aeid R and dilute to 1000 mL with water R.
slope (S) of the haemolysis curve at the approximate Gelatin barbital buffer solution Add 4 volumes of
infiexion point by segmenting the steepest section in suitable gelatin solution to 1 volume of barbital buffer stock solution
time intervals (for example, I'!.t = 1 min), and calculate S
and mix. Adjust to pH 7.3, if necessary, using 1 M sodium
between' adjacent intersection points, expressed as M per hydroxide or 1 M hydroehlorie acid. Maintain at 4 oc. Prepare
minute . The largest value for S serves as Sexp. In addition, fresh solutions daily.
determine the absorbance at the start of measurement (As )
by extrapolating the curve, which is almost linear and parallel
Stabilised sheep blood Colleet 1 volume of sheep blood
into 1 volume of citrate solution and mix. Store at 4 oC for
to the time axis within the first few minutes . Correct Sexp
using the expression: not less than 7 days and not more than 28 days. (Stabilised
sheep blood and sheep red blood cells are available from a
number of eommereial sourees .)
S' = S exp
As Haemolysin Antiserum against sheep red blood eells
prepared in rabbits. (Such antisera are available from a
number of eommereial sources.)
Calculate the arithmetic mean of the values of S' for each
preparation (test and reference solution). Calculate the index Guinea-pig complement Prepare a pool of serum from
of Fc function (lFc) from the expression: the blood of not fewer than 10 guinea-pigs. Separate the
serum from the clotted blood by eentrifugation at about
4 cC. Store the serum in small amounts below - 70 oc.
METHOD
Preparation of standardised 5 per cent sheep red blood
S' arithmetic mean of the corrected slope for the cell suspension Separate sheep red blood eells by
preparation to be examined, centrifuging an appropriate volume of stabilised sheep blood
S~ arithmetic mean of the corrected slope for the and wash the eells at least 3 times with gelatin barbital buffer
reference preparation, solution and prepare a 5 per eent V/V suspension in the
S~ arithmetic mean of the corrected slope for the same solution. Measure the eell density of the suspension as
complement control. follows: add 0.2 mL to 2.8 mL of water R and centrifuge the
Iysed solution for 5 min at 1000 g; the eell density is suitable
Calculate the index of Fc function for the preparation to be
if the absorbanee (2.2.25) of the supernatant liquid at
examined: the value is not less than that stated in the leafiet
541 nm is 0.62 ± 0.01. Correet the eell density by adding ,
accompanying the reference preparation.
gelatin barbital buffer solution aecording to the following
equation:
el. Test for anticomplementary activity of
irnmunoglobulin
VixA
(Ph. Eur. method 2.6.17) VI = 0.62
For the measurement of anticomplementary activity (ACA)
of irnmunoglobulin, a defined amount of test material
VI final adjusted volume;
(10 mg of immunoglobulin) is incubated with a defined
Vi the initial volume;
amount of guinea-pig complement (20 CHso ) and the
remaining complement is titrated; the anticomplementary
A absorbanee of the original suspension at 541 nm.
activity is expressed as the percentage consumption of The adjusted suspension eontains about 1 x 10 9 eells/mL.
complement relative to the complement control considered as Haemolysin titration Prepare haemolysin dilutions as
100 per cent. shown in Table 2.6.17.-1.
The haemolytic unit of complement activity (CH so) is the
amount of complement that, in the given reaction conditions,
will produce the Iysis of 2.5 x 10 8 out of a total of 5 x
10 8 optimally sensitised red blood cells.
Table 2.6.17.-1 The haemolysin titration is not valid unless the maximum
Required dilution Prepared using degree of haemolysis is 50 per cent to 70 per cent. If the
of haemolysin maximum degree of haemolysis is not in this range, repeat
Gelatin barbital the titration with more or less diluted complement solution.
Haemolysin
buffer solution Preparation of optimised sensitised sheep red blood
Volume Dilution Volume
celIs (haemolytic system) Prepare an appropriate volume
(mL) 0/--.) (mL) of diluted haemolysin containing 2 MHU/mL and an equal
7.5 0.65
volume of standardised 5 per cent sheep red blood cen
undiluted 0.1
suspension. Add the haemolysin dilution to the standardised
10 0.90 undiluted 0. 1 cell suspension and mix. Incubate at 37 oC for 15 min, store
75 1.80 7.5 0.2 at 2 oC to 8 oC and use within 6 h.
100 1.80 10 0.2
Titration of complement Prepare an appropriate dilution
of complement (for example 1/250) with gelatin barbital
150 1.00 75 1.0 buffer solution and perfortn the titration in duplicate as
200 1.00 100 1.0 shown in Table 2.6 .17.-2.
300 1.00 150 1.0
Table 2.6.17.-2
400 1.00 200 1.0
Tube number Volume of diluted complement Volume of gelatin barbital
600 1.00 300 1.0 (for example 1/250) buffer solution
(mL) (mL)
800 1.00 400 1.0
0.1 1.2
1200 1.00 600 1.0
2 0.2 l.l
1600 1.00 800 1.0
3 0.3 1.0
2400 1.00 1200 1.0
4 0.4 0.9
3200' 1.00 1600 1.0
5 0.5 0.8
4800' 1.00 2400 1.0
6 0.6 0.7
• discard 1.0 mL of the mixture.
7 0.7 0.6
8 0.8 0.5
Add 1.0 mL of 5 per cent sheep red blood cell suspension to
each tube of the haemolysin dilution series, starting at the 9 0.9 0.4
1/75 dilution, and mix. Incubate at 37 oC for 30 mino 10 1.0 0.3
Transfer 0.2 mL of each of these incubated mixtures to new
11 l.l 0.2
tubes and add 1.10 mL of gelatin barbital buffer s.olution and
0.2 mL of diluted guinea-pig complement (for example, 12 1.2 0.1
1/150). Perfortn this in duplicate. 3 tubes as cell 1.3
As the unhaemolysed cell control, prepare 3 tubes with control at O per
cent haemolysis
1.4 mL of gelatin barbital buffer solution and 0.1 mL of
3 tubes at 1.3 mL of water
5 per cent sheep red blood cen suspension. 100 per cent
As the fully haemolysed control, prepare 3 tubes with 1.4 mL haemolysis
of water R and 0.1 mL of 5 per cent sheep red cen
suspension. Add 0.2 mL of sensitised sheep red blood cells to each tube,
Incubate all tubes at 37 oC for 60 min and centrifuge at mix well and incubate at 37 oC for 60 mino Cool the tubes in
1000 g for 5 mino Measure the absorbance (2.2.25) of the an ice-bath and centrifuge at 1000 g for 5 mino Measure rhe
supematants at 541 nm and calculate the percentage degree absorbance of rhe supernatant liquid at 541 nm and calculate
of haemolysis in each tube using the following express ion: the degree of haemolysis (Y) using the following expression:
Ac absorbance of tubes 1 to 12;

Aa absorbance of tubes with haemolysin dilution; Ab mean absorbance of tubes with 100 per cent
Ab mean absorbance of the 3 tubes with full haemolysis; haemolysis;
Al mean absorbance of the 3 tubes with no haemolysis. Al mean absorbance of cell controls with O per cent
Plot the percentage degree of haemolysis as the ordinate haemolysis.
against the corresponding reciprocal value of the haemolysin Plot Y/(l - Y) as the abscissa against rhe amount of diluted
dilution as the abscissa on linear graph paper. Detertnine the complement in millilitres as the ordinate on log- Iog graph
optimal dilution of the haemolysin from the graph by papero Fit the best line to the points and detertnine the
inspection. Select a dilution such that further increase in the ordinate for the 50 per cent haemolytic complement dose
amount of haemolysin do es not cause appreciable change in where Y/ (l - Y) = 1.0. Calculate the activity in haemolytic
the degree of haemolysis. This dilution is defined as units (CH 501rnL) using the following expression:
1 minimal haemolytic unit (1 MHU) in 1.0 mL. The optimal
haemolytic haemolysin dilution for preparation of sensitised
sheep red blood cens contains 2 MHU/mL.
reciprocal value of the complement dilution; The International Unit is rhe activity contained in a stated
volume of diluted complement resulting in amount of the International Reference Preparation.
50 per cent haemolysis, in millilitres; The equivalence in International Units of the International
5 scaling factor to take account of the number of red Reference Preparation is stated by rhe World Health
blood celIs. Organization.
Human anti-D immunoglobulin BRP is calibrated in
The test is not valid unless the plot is a straight line between
International Units by comparison with the International
15 per cent and 85 per cent haemolysis and the slope is
Standard and intended for use in the assay of human anti-D
0.15 to 0.40, and preferably 0.18 to 0.30.
immunoglobulin.
Test for anticomplementary activity Prepare a
Use pooled D-positive red blood ceIls, coIlected not more
complement dilution having 100 CHsolmL by diluting
rhan 7 days earlier and suitably stored, obtained from not
titrated guinea-pig compIement with gelatin barbital buffer
fewer rhan 4 group O R¡R¡ donors. To a suitable volume of
solution. Depending on the immunoglobulin to be examined
the ceIls, previously washed 3 times with a 9 gIL solution of
and based on validation data, a pH adjustrnent to 7 may be
sodium chloride R, add an equal volume of bromelains
necessary. Prepare incubation mixtures as folIows for an
solution R, alIow to stand at 37 oC for 10 min, centrifuge,
immunoglobulin containing 50 mglrnL:
remove the supernatant liquid and wash 3 times with a 9 gIL
solution of sodium chloride R . Suspend 20 volumes of the red
Table 2.6.17.·3
blood celIs in a mixture of 15 volumes of inert semm,
Immunoglobulin Complement control
to be examined (in duplicate)
20 volumes of a 300 gIL solution of bovine albumin R and
Irnmunoglobulin (50 mg,!mL) 0.2 mL 45 volumes of a 9 gIL solution of sodium chloride R . Stand the
resulting suspension in iced water, stirring continuously.
Gelatin barbital buffer 0.6 mL 0.8 rnL
Using a calibrated automated dilutor, prepare suitable
Cornplement 0.2 mL 0.2 rnL dilutions of the preparatian to be examined and of the
reference preparation using as diluent a solution containing
5 giL of bovine albumin R and 9 gIL pf sodium chloride R.
Carry out the test on the immunoglobulin to be examined
and prepare ACA negative and positive controls using human Use a suitable apparatus for automatic continuous analysis.
immunoglobulin (AGA and molecular size) BRP, as indicated in The folIowing protocol is usualIy suitable: maintain the
the leafiet accompanying the reference preparation. Higher or temperature in the manifold, except for the incubation coils,
lower volumes of sample and of gelatin barbital buffer at 15.0 oc. Pump into the manifold of rhe apparatus the red
solution are added if the immunoglobulin concentration blood ceIl suspension at arate of 0.1 mUmin and a 3 gIL
varíes from 50 mglmL; for example, 0.47 mL of gelatin solution of methylcellulose 450 R at arate of 0.05 mUmin.
barbital buffer solution is added to 0.33 mL of Introduce rhe dilutions of rhe preparation to be examined
immunoglobulin containing 30 mglmL to give 0.8 mL. Close and the reference preparation at arate of 0.1 mUmin for
the tubes and incubate at 37 oC for 60 min. Add 0.2 mL of 2 min, folIowed by the diluent solution at arate of
each incubation mixture to 9.8 mL of gelatin barbital buffer 0.1 mUmin for 4 min before the next dilution is introduced.
solution to dilute the complement. Perform complement Introduce air at arate of 0.6 mUmin. Incubate at 37 oC for
titrations on each tube as descríbed aboye to determine the 18 min and rhen disperse rhe rouleaux by introducing at a
remaining complement activity (Table 2.6.17.-2). Calculate rate of l.6 mUmin a 9 gIL solution of sodium chloride R
the anticomplementary activity of the preparation to be containing a suitable wetting agent (for example, polysorbate
examined relative to the complement control considered as 20 R at a final concentration of 0.2 gIL) to prevent
100 per cent, using the folIowing expression: dismption of the bubble pattern. AIIow the agglutinates to
settle and decant twice, first at 0.4 mUmin and then at
a-b 0.6 mUmin. Lyse rhe unagglutinated red blood ceIls with a
- - x 100 solution containing 5 gIL of octoxinollO R, 0.2 gIL of
a
potassium ferricyanide R, 1 gIL of sodium hydrogen carbonate R
and 0.05 gIL of potassium cyanide R at arate of 2.5 mUmin.
a mean complement activity (CHsolmL) of complement
A lO-minute delay coil is introduced to aIlow for conversion
control;
of rhe haemoglobin. Continuously record the absorbance
b compl~ment activity (CHsolmL) of tested sample.
(2.2.25) ofthe haemolysate at a wavelength between 540 nm
The test is not valid unless: and 550 nm. Determine the range of antibody concentrations
- the anticomplementary activities found for ACA negative over which there is a linear relationship between the
control and ACA positive control are within the limits concentration and rhe resultant change in absorbance (M).
stated in the leafiet accompanying the reference From the results, prepare a standard curve and use rhe linear
preparation; portion of rhe curve to determine rhe activity of the
- the mean complement activity of complement control (a) preparation to be examined.
is in the range 80 CHsolrnL to 120 CHsolmL. Calculate rhe potency of the preparation to be examined
using rhe usual statistical methods (5.3).
e3. Assay ofhuman anti-D immunoglobulin MethodB
The potency of human anti-D immunoglobulin is determined
MethodA by competitive enzyme-linked immunoassay on erythrocyte·
The potency of human anti-D immunoglobulin is determined coated microtitre plates. The method is based on the
by comparíng the quantity necessary to produce agglutination competitive binding between a polyclonal anti-D
of D-positive red blood ceIls wirh rhe quantity of a reference immunoglobulin preparation and a biotinylated monoclonal
preparation, calibrated in International Units, required ro anti-D antibody directed against a D-antigen-specific epitope .
produce rhe same effect.
V -A416 Appendix XIV J 2014
The activity of the preparation to be examined is compared J'viETHOD

with a reference preparation calibrated in International Units . Prepare a 0.1 per cent V/V suspension of papain-treated red
The Intemational Unit is the activity of a stated amount of blood ceIls in cold ceIl-fixation buffer. Pipette 50 IlL into
Intemational Reference Preparation. The equivalence in each weIl of the fiat-bottomed microtitre plate .
Intemational Units of the Intemational Reference Centrifuge the plate at 350 g for 3 min, preferably at 4 oC,
Preparation is stated by the WorId Health Organization. Without removing the supernatant, gently add 100 IlL of
Human anti-D immunoglobulin BRP is calibrated in glutaraldehyde solution to each weIl and leave for 10 mino
Intemational Units by comparison with the Intemational Drain the weIls by quickly inverting the plate and wash
Standard and intended for use in the assay of human anti-D 3 times with 250-300 pL of PBS. This may be done
immunoglobulin. manuaIly or using a suitable automated plate washer. Either
MATERIALS carry out the assay as described below, or store the plate at
Reagents not specified are of analytical grade. 4 oC after draining off the PBS and adding 100 ~lL of ceIl-
fixation buffer per well and sealing with plastic film . Plates
PBS (Phosphate-buffered saline) Dissolve 8.0 g of
can be stored at 4 oC for up to 1 month.
sodium ehlonde R, 0.76 g of anhydrous disodiwn hydrogen
phosphate R , 0.2 g of potassium ehloride R, 0.2 g of potassiwn Test solutions For freeze-dried preparations, reconstitute
dihydrogen phosphate R and 0.2 g of sodium azide R in water R as stated on the label. Prepare 4 independent replica tes of 5
and dilute to 1000 mL with the same solvent. serial 2-fold dilutions starting with 30 IU/mL in PBS
containing 10 giL of bovine albumin R. If necessary, adjust
TBS (Tris-buffered saline) Dissolve 8.0 g of sodium
the starting dilution to obtain responses faIling in the linear
ehlonde R and 0.6 g of tns(hydroxymethyl)aminomethane R in
portion of the dose-response curve.
water R. Adjust to pH 7.2 with 1 M hydroehlone acid and
dilute to 1000 mL with water R. Reference soiutions Reconstitute the reference
preparation according to instructions. Prepare 4 independent
Papain solution Prepare a solution by stirring 1 g of
replica tes of 5 serial 2-fold dilutions starting with 30 IU/mL
papain R at 37 oC for 30 min in 10 mL of 0.067 M
in PBS containing 10 giL of bovine albumin R.
phosphate buffer solution pH 5.4 R , centrifuge at 10 000 g for
5 min and filter through a membrane filter (nominal pore Using U- or V-bottomed microtitre plates, add 35 ¡.tL of each
size 0.22 11m). To activate, combine 1 mL of the filtra te with of the dilutions of the test solution or reference solution to
1 mL of a 48.44 gIL solution of L-eysteine R and 1 mL of a each of a series of weIls. To each weIl add 35 ¡.tL of
3.72 gIL solution of sodium edetate R and dilute to 10 mL biotinylated Brad-5 at 250 nglmL.
with 0.067 M phosphate buffer so/ution pH 5.4 R . Freeze in Empty the weIls of the red ceIl-coated plate by inverting and
aliquots at -20 oC or below. draining on a paper towel. Add 250 ¡.tL of PBS containing
Red blood cells Use pooled D-positive red blood ceIls 20 gIL of bovine albumin R and leave at room temperature for
obtained fro~ not fewer than 3 group O RzRz donors. Wash 30 mino
the ceIls 4 times with PBS. Centrifuge the ceIls at 1800 g for Empty the weIls of the red ceIl-coated plate by inverting and
5 min, mix a suitable volume of prewarmed packed ceIls draining on a paper towel and transfer 50 ¡.tL from each of
with a suitable volume of prewarmed papain solution the dilutions of the test solution or reference solution
(2 volumes to 1 volume has been found suitable) and containing biotinylated Brad-5 into the wells, Use 50 !1L of
incubate at 37 oC for 10 mino Wash the ceIls 4 times with PBS containing 10 gIL of bovine albumin R as negative
PBS. Store at 4 oC in an appropriate stabiliser for up to control. Seal the plate with plastic film and incubate at room
1 week. temperature for 1 h.
Biotinylated Brad-5 Use according to instructions. Remove the liquid from the weIls of the red ceIl-coated plate
Alkaline phosphatase-conjugated avidin/streptavidin and wash 3 times with 250-300 IlL of TBS.
reagent Preferably modified to combine high specific Dilute the alkaline phosphatase-conjugated avidinlstreptavidin
activity with low non-specific binding. Use according to reagent in TBS containing 10 gIL of bovine albumin R and
instructions. add 50 JlL to each well. Incubate for 30 min at room
Substrate solution Use para-nitrophenyl phosphate temperature.
according to instructions. Remove the liquid from the weIls of the red ceIl-coated plate
Cell fixation buffer Dissolve 18.02 g of glueose R, 4.09 g and wash 3 times with 250-300 JlL of TBS,
of sodium ehlonde R, 1.24 g of bone acid R , 10.29 g of sodium Add 100 ¡.tL of substrate solution to each of the weIls and
eitrate R 'and 0.74 g of sodium edetate R in water R. Adjust to incubate at room temperature for 10 min in the dark.
pH 7.2-7.3 using 1 M sodium hydroxide or 1 M hydroehlone To stop the reaction, add 50 ¡.tL of 3 M sodium hydroxide to
acid, and dilute to 1000 mL with water R, Use directly from each of the weIls,
storage at 4 oc, Measure the absorbances at 405 nm and substract the
Glutaraldehyde solution Immediately before use, add negative control reading. Use the absorbance values in the
750 !1L of a 250 gIL solution of glutaraldehyde R to 50 mL of linear range of the titration curve to estimate the potency of
cold PBS , the preparation to be examined by the usual statistical
Microtitre piates Plates to be coated with red blood cells methods (5.3).
are fiat-bottomed polystyrene plates with surface properties Method C
optimised for enzyme immunoassay and high protein-binding The potency of human anti-D immunoglobulin is determined
capacity. Plates used to prepare immunoglobulin dilutions by fiow cytometry in a microtitre plate formato The method
are U- or V-bottomed polystyrene or poly(vinyl chloride) is based on the specific binding between anti-D
plates. immunoglobulin and D-positive red blood ceIls. The activity
of the preparation to be examined is compared with a
reference preparation calibrated in Intemational Units.
2014 Appendix XIV J V -A41 7
The Intemational Unit is the activity of a stated amount of Seal with plastic film and incubate, protected from light, at
Intemational Reference Preparation. The equivalence in room temperature for 20 mino
Intemational Units of the Intemational Reference preparation Centrifuge the plates at 50 g for 3 min, discard the
is stated by the World Health Organization. supematant and wash the cells with 200-250 ¡.tL of PES-BSA
Human anti-D immunoglobulin BRP is calibrated in solution. Repeat this at least once.
Intemational Units by comparison with the International Centrifuge the plates at 50 g for 3 min, resuspend the cells
Standard and intended for use in the assay of human anti-D into 200-250 ¡.tL of PBS. Transfer the cell suspension into a
immunoglobulin. tube suitable for the flow-cytometry equipment available and
MATERIALS further dilute by adding PBS to allow a suitable flow rateo
Reagents not specified are of analytical grade. Proceed irnmediately with measurement of the median
PBS Dissolve 8.0 g of sodium ehloride R, 0.76 g of disodium fluorescence intensity in a flow cytometer. Record at least
hydrogen phosphate R, 0.2 g of potassium ehloride R and 0.2 g 10000 events without gating but excluding debris.
of potassium dihydrogen phosphate R in water R and dilute to Use the median fluorescence intensity in the linear range of
1000 mL with the same solvento the dose-response curve to estimate the potency of the
PBS-BSA solution PBS containing 10.0 giL of bovine preparation to be examined by the usual statistical methods
albumin R. (5.3).
Red blood cells Use D-positive red blood cells obtained
C4. Test for anti-D antibodies in human
from a group O R¡R¡ donor within 2 weeks of collection.
immunoglobulin
Store if necessary in an appropriate stabiliser at 4 oc. Wash
the ceJls at least twice with PBS-BSA solution and prepare a (Ph. Eur. method 2.6.26)
suspension containing 1 x 104 ceJls per microlitre but not MATERIALS
more than 5 x 104 ceJls per microlitre in PBS-BSA solution. Phosphate-buffered saline (PBS) Dissolve 8.0 g of
Use D-negative red blood cells obtained from a group O rr sodium ehloride R, 0.76 g of anhydrous disodium hydrogen
donor and prepared similarly. phosphate R, 0.2 g of potassium ehloride R and 0.2 g of
Secondary antibody Use a suitable fluorescent dye- potassium dihydrogen phosphate R in water R and dilute to
conjugated anti-IgG antibody fragment specific for human 1000 mL with the same solvent. If the solution has to be
IgG or parts of it. Store and use according to the kept for several days, 0.2 g of sodium azide R may be added
manufacturer's instructions. in order to avoid microbial contamination.
Microtitres plates Use flat-bottomed plates without PBS-BSA solution PES containing 2 gIL of bovine
surface treatment for enzyme irnmunoassays. albumin R (Cohn Fraction V, for EUSA) . Store the solution
METHOD
at 2-8 oC but allow it to reach 19-25 oC before use.
Test solutions For freeze-dried preparations, reconstitute Papain solution Use serological-grade papain from a
as stated on the labe!' Prepare at least 3 independent commercial source, the activity of which has been validated.
replicates of at least 3 serial 1.5- or 2-fold dilutions starting Red blood cells Use pooled D-positive red blood cells
with a concentration in the range of 1.2-0.15 IU/mL using from not fewer than 3 donors, preferably of group OR2 R 2 •
PBS/BSA solution as diluent. If necessary, adjust the starting D-positive red blood cells may also be obtained from OR¡R¡
dilution to obtain responses falling in the linear portion of or OR¡R2 donors. Mixing phenotypes has not been tested
the dose-response curve. and is therefore not recommended.
Reference solutions Reconstitute the reference Use pooled D-negative red blood cells, preferably from
preparation according to instructions. Prepare at least 3 3 donors of group Orr. When only 1 donor of group Orr is
independent replicates of at least 3 serial 1.5- or 2-fold available, D-negative red blood cells from only 1 donor may
dilutions starting with a concentration in the range of be used.
1.2-0.15 IU/mL using PBS-BSA solution as diluent. Wash the cells 4 times with PBS or until the supematant is
If necessary, adjust the starting dilution to obtain responses clear. Each wash consists of suspending the cells in a
falling in the linear portion of the dose-response curve. minimum of 2 volumes of PBS, centrifuging the cells at 1800
Distribute 50 ~lL of the D-positive red blood cells into each g for 5 min to pack, and discarding the supematant. Treat
well of a microtitre plateo Add 50 ¡.tL of each of the dilutions the packed cells with papain solution according to the
of the test solution or reference solution to each of a series of manufacturer's instructions and wash the cells 4 times with
wells. Use 50 ¡.tL of PBS-BSA solution as negative contro!. PBS.
Distribute 50 ¡.tL of the D-negative red blood cells into Red blood cells may be stored for not more than 1 week in a
4 wells of the same microtitre plate and add 50 ¡.tL of the preservative solution at 2-8 oc. A preparation of the
lowest dilution of the test preparation. To monitor spurious following composition is appropriate:
reactions, distribute 50 ¡.tL of the D-positive red blood cells Trisodium citrate 8 gIL
into 4 wells of the same microtitre plate and add 50 ¡.tL of D-glucose 20 giL
PBS-BSA solution. Seal with plastic film and incubate at Citric acid 0.5 giL
37 oC for 40 min. 4.2 giL
Sodium chloride
Centrifuge the plates at 50 g for 3 min, discard the Inosine 0.938 giL
supernatant and wash the cells with 200-250 ¡.tL of PBS-BSA Adenosine triphosphate (ATP) 0.4 giL
solution. Repeat this at least once. Chloramphenicol 0.34 giL
Centrifuge the plates at 50 g for 3 min, discard the Neomycin sulfate 0.1 giL
supematant and add 50 ¡.tL of the secondary antibody diluted If using stored cells, wash the cells at least twice in PES or
with PBS-BSA solution to a suitable protein concentration. until the supematant is clear before proceeding.
Microtitre plates Use V-bottomed rigid microtitre plates. The titre of the preparation 10 be examined must not be
Reference standards Immunoglobulin (anti-D antibodies greater than the titre of the positive control when both
test) BRP and Immunoglobulin (anti-D antibodies test negative preparations are titrated from 25 gIL.
control) BRP are suitable for use as the positive control and
negative control, respectively. C5. Anti-A and anti-B haemagglutinins
(Ph. Eur. method 2.6.20 )
METHOD
The test described in this chapter is performed at room METHOD A: INDlRECT METHOD
temperature on the positive control solutions, the negative Prepare in duplicate serial dilutions of the preparation 10 be
control solutions and the test solutions at the same time and examined in a 9 gIL solution of sodium ehlonde R. To each
under identical conditions. dilution of 1 series add an equal volume of a 5 per cent V/V
Reference solutions Reconstitute the positive control and suspension of group A¡ red blood ceIls previously washed
the negative control according to the instructions. 3 times with the sodium chloride solution. To each dilution
The immunoglobulin G (IgG) concentration is 50 gIL in of the other series add an equal volume of a 5 per cent V/V
each of the reconstituted preparations. Make a 2-fold suspension of group B red blood ceIls previously washed
dilution of each reconstituted preparation with PBS-BSA 3 times with the sodium chIoride solution. Incubate the
solution to obtain solutions containing IgG at 25 giL. suspensions at 37 oC for 30 min then wash the ceIls 3 times
Prepare 7 further serial 2-fold dilutions of each preparation with the sodium chloride solution. Leave the ceIls in contact
using PBS-BSA solution 10 extend the dilution range 10 with a polyvalent anti-human globulin reagent for 30 mino
1/256 (0.195 gIL IgG). Add 20 IlL of each dilution of each Without centrifuging, examine each suspension for
preparation in duplicate 10 the microtitre plate. agglutination under a microscope.
Test solutions Dilute the preparation to be examined with METHOD B: DIRECT METHOD
PBS-BSA solution to obtain a starting IgG concentration of MATERIALS
25 giL. For 50 gIL preparations, this is a 2-fold dilution; Phosphate-bufJered saline (PBS) Dissolve 8.0 g of
adjust the dilution factor accordingly for preparations with an sodium ehloride R, 0.76 g of anhydrous disodium hydrogen
IgG concentration other than 50 gIL to obtain a starting phosphate R, 0.2 g of potassium ehloride R and 0.2 g of
concentration of 25 gIL for testing. This 25 gIL solution is . potassium dihydrogen phosphate R in water R and dilute to
assigned a nominal 2-fold dilution factor for comparison with 1000 mL with the same solvent. If the solution has 10 be
the reference solutions, even if this does not refiect the true kept for several days, 0.2 g of sodium azide R may be added
dilution factor used 10 achieve 25 gIL. Prepare 7 further in order 10 avoid microbial contamination.
serial 2-fold dilutions of the preparation using PBS-BSA PBS-BSA solution PBS containing 2 gIL of bovine
solution 10 extend the nominal dilution range 10 1/256 albumin R (Cohn Fraction V, for EUSA). S10re the solution
(0.195 giL IgG) for comparison with the reference at 2-8 oC but aIlow it to reach 19-25°C before use.
preparations over the same IgG concentration range.
Papain solution Use serological-grade papain from a
Add 20 IlL of each dilution in duplicate to the microtitre
commercial source, the activity of which has been validated.
plate.
Red blood cells Use pooled D-negative Al (Alrr),
Prepare 3 per cent V/V suspensions of papain-treated
D-negative B (Brr) and D-negative O (Orr) red blood ceIls
D-positive (preferably OR2 R2 , but OR¡R¡ or OR¡R2 may
from preferably 3 donors. When Immunoglobulin for anti-A
also be used) and D-negative (Orr) red blood ceJIs in PBS-
and anti-B antibodies limit test BRP is used, 3 donors are 10
BSA solution. Add 20 IlL of D-positive red blood ceJIs to 1
be used. A2 red blood ceIls are not recommended as they
dilution series of each of the preparation 10 be examined, the
give weaker reactions.
positive control and the negative control, and 20 IlL of
D-negative red blood ceJIs to the other dilution series. Wash the ceIls 4 times with PBS or until the supernatant is
Mix by shaking the plate on a shaker for 10 s (or until the cIear. Each wash consists of suspending the ceIls in a
ceIls are resuspended). minimum of 2 volumes of PBS, centrifuging the ceIls at 1800
g for 5 min to pack, and discarding the supernatant. Treat
Centrifuge the plate at .80 g at room temperature for 1 min
the packed ceIls with papain solution according to the
to pack the celIs. Place the plate at an angle of approximately
manufacturer's instructions and wash the ceIls 4 times with
70°. Read after 4-5 min or when the negative controls
PBS.
(D-negative red blood ceIls and negative control solution)
have streamed. A ceIl button at the bottom of the weIl Red blood ceIls may be sto red for not more than 1 week in a
indica tes a positive result. A stream of ceIls represents a preservative solution at 2-8 oC. A preparation of the
negative result. folIowing composition is appropriate:
Record the endpoint titre as the reciprocal of the highest Trisodium citrate 8 giL
dilution that gives rise 10 a positive resulto D-glucose 20 giL
Citric acid 0.5 gIL
The positive control has a nominal titre of 8 and the negative
Sodium chIoride 4.2 giL
controls (D-negative red blood ceIls and negative control
Inosine 0.938 gIL
solution) must not show agglutination at the starting dilution
Adenosine triphosphate (ATP) 0.4 gIL
of 1 in 2. Users must validate their own test conditions, and
ChIoramphenicol 0.34 giL
investigate their assay conditions and reagents in the event of
Neomycin sulphate 0.1 giL
results being significantly different from those expected.
Failure to obtain negative reactions with the negative control s If using s10red ceIls, wash the ceIls at least twice in PBS or
may indicate that, for example, insufficient time has elapsed until the supernatant is cIear before proceeding.
for the ceIls 10 stream, or that reagents have been used Microtitre plates Use V-bottomed rigid microtitre plates.
directly from cold s1Orage. Reference standards Immunoglobulin (anti-A, anti-B
antibodies test positive control) BRP and bnmunoglobulin (anti-
2014 Appendix XIV J V -A41 9
A, anti-B antibodies test negative control) BRP are suitable for from those expected. Failure to obtain negative reactions
use as the positive control and negative control, respectively, with the negative controls may indica te that, for example,
and should be used as guides for operators establishing and insufficient time has elapsed for the cells to stream, or that
performing the direct method for anti-A and anti-B reagents have been used directly from cold storage.
haemagglutinins. If the anti-A or anti-B titre of the preparation to be examined
Immunoglobulin lor anti-A and anti-B antibodies limit test BRP is greater than the titre of the positive control when both
defines the recommended maximum limits permissible for preparations are titrated from 25 gIL, the test preparation is
batches of human immunoglobulin and must be used only to be compared with Immunoglobulin lor anti-A and anti-B
for comparison with batches of human immunoglobulin that antibodies limit test BRP.
have higher titres than the positive control. The maxirnum allowable titre is 64 when the preparations
METHOD are titrated from 25 giL.
The test described in this chapter is performed at room
temperature on the positive control solutions, the negative Other blood-related components
control solutions and the test solutions at the same time and DI. Assay ofhuman ex-l-proteinase inhibitor
under identical conditions. Whenever necessary, a further test
is performed with Immunoglobulin lor anti-A and anti-B
antibodies limit test BRP.
Human ex-1-proteinase inhibitor (al so known as ex-1-
antitrypsin or ex-1-antiproteinase) content is determined by
Reference solutions Reconstitute the, positive control and comparing the ability of the preparation to be examined to
the negative control according to the instructions. inactivate the serine protease elastase (porcine pancreatic
The immunoglobulin G (IgG) concentration is 50 gIL in elastase or human neutrophil elastase) with the same ability
each of the reconstituted preparations. Make a 2-fold of a reference standard of human ex-1-proteinase inhibitor
dilution of each reconstituted preparation with PBS-BSA calibrated in milligrams of active (functional) ex-1-proteinase
solution to obtain solutions containing IgG at 25 giL. inhibitor. Varying quantities of the preparation to be
Prepare 7 further serial 2-fold dilutions of each preparation examined are mixed with a given quantity of el astas e and the
using PBS-BSA solution to extend the dilution range to remaining el astas e activity is determined using a suitable
1/256 (0.195 gIL IgG). Add 20 ¡.¡L of each dilution of each chromogenic substrate. The method described below is given
preparation in triplicate to the microtitre plate. as an example.
Test solutions Dilute the preparation to be examined with
PBS-BSA solution to obtain a starting IgG concentration of Reagents
25 giL. For 50 gIL preparations, this is a 2-fold dilution; Tris-albumin buffer solution Dissolve 24.23 g of
adjust the dilution factor accordingly for preparations with an trometamol R in water R, adjust to pH 8.0 ± 0.3 using
IgG concentration other than 50 gIL to obtain a starting hydrochloric acid Rl and dilute to 1000 mL with water R.
concentration of 25 giL for testing. This 25 gIL solution is To 100 mL of this solution add 0.5 mL of a 20 per cent
assigned a nominal 2-fold dilution factor for comparison with solution of human albumin R or bovine albumin R.
the reference solutions, even if this does not refiect the true Buffer solution containing human or bovine albumin must be
dilution factor used to achieve 25 gIL. Prepare 7 further prepared fresh on the day of its use; otherwise, it can be
serial 2-fold dilutions of the preparation using PBS-BSA conserved by sterile filtration (0.2 ¡.¡m) and stored at 2-8 oC
solution to extend the nominal dilution range to 1/256 for up to 2 weeks.
(0.195 giL IgG) for comparison with the reference Method
preparations over the same IgG concentration range.
Prepare 2 series of 4 or 5 dilutions in an appropriate human
Add 20 ~lL of each dilution in triplicate to the microtitre
ex-1-proteinase inhibitor concentration range, for both the
plate.
preparation to be examined and the reference standard, using
Prepare 3 per cent V/V suspensions of papain-treated the tris-albumin buffer solution.
D-negative Al' B and O red blood cells in PBS/BSA
Transfer 50 ¡.¡L of the reference solution dilutions into the
solution. Add 20 ~lL of D-negative Al' B and O red blood
wells of a microtitre plate and to each well, add 150 J.lL of a
cells respectively to the 1SI, the 2nd and the 3rd dilution series
porcine pancreatic elastase solution diluted to an appropriate
of each of the preparation to be examined, the positive
concentration with the tris-albumin buffer solution. Incubate
control and the negative control. Mix by shaking the plate on
for a defined period of time, 3-10 min, at room temperature.
a shaker for lOs (or until the cells are resuspended).
Since the activity of the solutions of the different porcine
Centrifuge the plate at 80 g at room temperature for 1 min pancreatic elastases may vary, the concentration of elastase
to pack the cells. Place the plate at an angle of approximately can be adjusted by evaluation of blank values containing
70°. Read after 4-5 min or when the negative controls el astas e but no human ex-1-proteinase inhibitor, to exhibit a
(D-negative O red blood cells and negative control solution) suitable change of absorbance at 405 nm under the actual
have streamed. A cell button at the bottom of the well assay conditions.
indica tes a positive resultoA stream of cells represents a
Add to each well 100 J.lL of a solution of chromogenic
negative resulto
substrate N-succinyl-tri-L-alanyl 4-p-nitroanilide (Suc-Ala-
Record the endpoint titre as the reciprocal of the highest Ala-Ala-pNA), reconstituted in dimethyl sulfoxide R to give a
dilution that gives rise to a positive result. solution containing 4.5 mglmL, then further diluted with the
The positive control has nominal anti-A and anti-B titres of tris-albumin buffer solution to a concentration of
32 (range 32-64 for anti-A; range 16-32 for anti-B) and the 0.45 rriglmL. Immediately start measurement of the change
negative controls (D-negative O red blood cells and negative in absorbance (2.2.25) at 405 nm using a microtitre plate
control solution) must not show agglutination at the starting reader, continuing the measurement for at least 5 min.
dilution of 1 in 2. Users must validate their own test Calculate the rate of change of absorbance (M/min).
conditions, and investigate their assay conditions and Altematively, an end-point assay may be used by stopping
reagents in the event of results being significantly different the reaction with acetic acid and measuring the absorbance at
V-A420 Appendix XIV K 2014
405 run. If the assay is performed in test tubes using serological assay of the tetanus vaccine (adsorbed) (2.7.8)
spectrophotometers for monitoring the change in absorbance when the common immunisation conditions for the
at 405 nm, the volumes of reagent solutions are changed diphtheria and the tetanus components (for example, doses,
proportionally. duration) have been demonstrated to be valid for the
The rate of change of absorbance (M/min) is inversely combined vaccine.
proportional to human cx-l-proteinase inhibitor activity. The design of the assays described below uses multiple
Check the validity of the assay and calculate the potency of dilutions for the test and reference preparations. Once the
the test preparation by the usual statistical methods (5.3). analyst has sufficient experience with this method for a given
vaccine, it is possible to apply a simplified model such as a
single dilution for both test and reference preparations. Such
a model enables the analyst to determine whether the
K. Immunological Products potency of the test preparation is significantly higher than the
minimum required, but does not give information on
1. Assay of diphtheria vaccine (adsorbed) linearity, parallelism and the dose-response curve.
(Ph. Eur. method 2.7.6. An alternative in vivo method (Method The simplified model allows for a considerable reduction in
B) in which the potency is determined by comparing the dose the number of animals required and must be considered by
necessary to protect guinea-pigs against the lethal effect 01 a each analyst in accordance with the provisions of the
subcutaneous il'yection 01 diphtheria toxin with the dose 01 a European Convention for the protection of vertebrate
relerence preparation calibrated in International Units necessary to animals used for experimental and other scientific purposes.
give the same protection isalso described in the European Where a single-dilution assay is used, production and test
Pharmacopoeia.) consistency over time are monitored via suitable indicators
The potency of diphtheria vaccine is determined by and by carrying out a full multiple-dilution assay periodically,
administration of the vaccine to guinea-pigs followed either for example every 2 years. For serological assays, suitable
by challenge with diphtheria toxin (method A) or by indicators to monitor test consistency are:
determination of the titre of antibodies against diphtheria - the mean and standard deviation of relative antitoxin titres
toxin or toxoid in the' serum of guinea-pigs (method C). or scores of the serum samples obtained after
In both cases, the potency of the vaccine is calculated by administration of a fixed dose of the vaccine reference
comparison with a reference preparation, calibrated in preparation;
Intemational Units.
- the antitoxin titres or scores of run controls (positive and
The Intemational Unit is the activity contained in a stated negative serum samples);
amount of the Intemational Standard, which consists of a
- the ratio of antitoxin titres or scores for the positive serum
quantity of diphtheria toxoid adsorbed on aluminium
control to the serum samples corresponding to the
hydroxide. The equivalence in Intemational Units of the
reference vaccine.
Intemational Standard is stated by the World Health
Organisation (WHO). Method A: Intradermal challenge test in guinea pigs
Diphtheria vaccine (adsorbed) BRP is suitable for use as a Selection and distribution ofthe test animals Use in
reference preparation. the test healthy, white guinea-pigs from the same stock and
The method chosen for the assay of diphtheria vaccine of a size suitable for the prescribed number of challenge
(adsorbed) depends -on the intended purpose. Method A is sites, the difference in body mass between the heaviest and
used: the lightest animal being not greater than 100 g. Use guinea-
pigs of the same sex or with males and females equally
1. during development of a vaccine, to assay batches
distributed between the groups. Distribute the guinea-pigs in
produced to validate the production;
not fewer than 6 equal groups; use groups containing a
2. wherever revalidation is needed following a significant number of animals sufficient to obtain results that fulfil the
change in the manufacturing process. requirements for a valid assay prescribed below. If the
Method A may also be used for the routine assay of batches challenge toxin to be used has not been shown to be stable
of vaccine, but in the interests of animal welfare, method C or has not been adequately standardised, inc1ude 5 guinea-
is used wherever -possible. pigs as unvaccinated controls.
Method C may be used, except as specified under 1 and 2 Selection of the challenge toxin Se1ect a preparation of
aboye, after verification of the suitability of the method for diphtheria toxin containing 67 to 133 Ir/ lOO in 1 Lf and
the product. For this purpose, a suitable number of batches 25 000 to 50 000 minimal reacting doses for guinea-pig skin
(usually 3) are assayed by method C and method A. Where in 1 Lf. If the challenge toxin preparation has been shown to
different vaccines (monovalent or combinations) are prepared be stable, it is not necessary to verify the activity for every
from diphtheria toxoid of the same origin, and with assay.
comparable levels (expressed in Lli'mL) of the same Preparation of the challenge toxin solution
diphtheria roxoid, suitability demonstrated for the Irnmediately before use, dilute the challenge toxin with a
combination with the highest number of components can be suitable diluent to obtain a challenge toxin solution
assumed ro be valid for combinations with fewer components containing about 0.0512 Lf in 0.2 mL. Prepare from this a
and for monovalent vaccines. Any combinations containing a further series of 5 four-fold dilutions containing about
whole-cell pertussis component or containing haemophilus 0.0128, 0.0032, 0.0008, 0.0002 and 0.00005 Lf in 0.2 mL.
type b conjugate vaccine with diphtheria toxoid or CRM 197
Dilution of the test and reference preparations U sing a
diphtheria protein as carríer in the same vial must always be
9 gIL solution of sodium chloride R, prepare dilutions of the
assessed separately.
vaccine to be examined and of the reference preparation,
For combinations containing diphtheria and tetanus such that for each, the dilutions form a series differing by
components, the serological assay (method C) can be not more than 2.5-fold steps and in which the intermediate
performed with the same group of animals used for the
2014 Appendix XIV K. V-A421
dilutions, when injected subcutaneously at adose of 1.0 mL Dilution ofthe test and reference preparations Using a
per guinea-pig, will result in an intradermal score of 9 gIL solution of sodium chloride R as diluent, prepare serial
approximately 3 when the animals are challenged. dilutions of the vaccine to be examined and the reference
Immunisation and challenge Allocate the dilutions, 1 to preparation; series differing by 2.5- to 5-fold steps have been
each of the groups of guinea-pigs, and inject subcutaneously found to be suitable. Use not fewer than 3 dilutions within
1.0 mL of each dilution into each guinea-pig in the group to the range of, for example, 0.5-16 IU/mL for the reference
which that dilution is allocated. After 28 days, shave both vaccine and within the range of, for example, 1:2 to 1: 125
flanks of each guinea-pig and inject 0.2 mL of each of the for the vaccine to be examined. Use the dilutions for
6 toxin dilutions intradermally into 6 separate sites on each immunisation preferably within 1 h of preparation. AlIocate
of the vaccinated guinea-pigs in such a way as to minimise 1 dilution to each group of guinea-pigs.
interference between adjacent sites. Immunisation Inject subcutaneously to each guinea-pig
Determination of the activity of the challenge toxin If 1.0 mL of the dilution allocated to its group.
necessary, inject the unvaccinated control animals with Blood sampling 35-42 days after irnmunisation, take a
dilutions containing 80, 40, 20, 10 and 5 x 10-6 Lf of the blood sample from each vaccinated and control guinea-pig
challenge toxin. using a suitable method.
Reading and interpretation of results Examine all Preparation of serum samples Avoid frequent freezing
injection sites 48 h after injection of the challenge toxin and and thawing of serum samples. To avoid microbial
record the inciden ce of specific diphtheria erythema. Record contamination, it is preferable to carry out manipulations in
also the number of sites free from such reactions as the a laminar-flow cabinet.
intra-dermal challenge score. Tabulate the intradermal Determination of antibody titre Determine the relative
challenge scores for all the animals receiving the same antibody titre or score of each serum sample by a suitable
dilution of vaccine and use those data with a suitable immunochemical method (2.7.1) . The methods shown below
transformation, such as (score)2 or arcsin ((score/6i), to (enzyme-linked immunosorbent assay (ELISA) and Vero cell
obtain an estimate of the relative potency for each of the test assay) have been found to be suitable.
preparations by parallel-line quantitative analysis. Calculation of potency Calculate the potency of the
Requirements for a valid assay The test is not valid vaccine to be examined in International Units relative to the
unless: reference preparation, using the usual statistical methods (for
- for both the vaccine to be examined and the reference example, 5.3) .
preparation, the meán score obtained at the lowest dose Requirements for a valid assay The test is not valid
level is les s than 3 and the mean score at the highest dose unless:
level is more than 3; - the confidence limits (P = 0.95) are not less than
- where applicable, the toxin dilution that contains 40 x 50 per cent and not more than 200 per cent of the
10-6 Lf gives a positive erythema in at least 80 per cent of estimated potency;
the control guinea-pigs and the dilution containing 20 x - the statistical analysis shows a significant slope and no
10-6 Lf gives a positive erythema in less than 80 per cent deviation from linearity and parallelism of the dose-
of the guinea-pigs (if these criteria are not met 'a different response curves (chapter 5.3 describes possible alternatives
toxin has to be selected); if significant deviations are observed).
- the confidence limits (P = 0.95) are not less than The test may be repeated but when more than 1 test is
50 per cent and not more than 200 per cent of the performed the results of all valid tests must be combined in
estimated potency; the estimate of potency.
- the statistical analysis shows no deviation from linearity The following section is published for information.
and parallelism.
The test may be repeated but when more than 1 test is Assay of diphtheria vaccine (adsorbed): guidelines
performed the results of all valid tests must be combined in Method C. Determination of antibodies in guinea pigs
the estimate of potency.
Preparation of serum samples For the preparation of
Method C. Determination of antibodies in guinea pigs serum samples, the following technique has been found to be
Selection and distribution ofthe test animals Use in suitable. Invert the tubes containing blood samples 6 times
the test healthy guinea-pigs [rom the same stock, each and allow to stand at 37 oC for 2 h, then at 4 oC for 2 h .
weighing 250-350 g. Use guinea-pigs of the same sex or with Centrifuge at room temperature at 800 g for 20 mino
males and females equally distributed between the groups. Transfer the serum to sterile tubes and store at a
Distribute the guinea-pigs in not fewer than 6 equal groups; temperature below - 20 oc. At least a 40 per cent yield of
use groups containing a number of animals sufficient to serum is obtained by this procedure.
obtain results that fulfil the requirements for a valid assay Determination of antibody titre The ELISA and Vero
prescribed below. Use a further group of non-vaccinated cell assays shown below are given as examples of
guinea-pigs of the same origin to provide a negative serum immunochemical methods that have been found to be
control. If test consistency has been demonstrated, a suitable for the determination of antibody titre.
reference negative serum control may be used. Determination of antibody titre in guinea-pig serum by
Reference preparation Use a suitable reference enzyme-linked immunosorbent assay (ELISA)
preparation such as diphtheria vaccine (adsorbed) BRP or a Dilutions of test and reference sera are made on ELISA
batch of vaccine shown to be effective in clinical studies, or a plates coated with diphtheria toxoid. A positive guinea-pig
batch representative thereof, and which has been calibrated serum control and a negative guinea-pig serum control are
in International Units with reference to diphtheria vaccine included on each plate to monitor the assay performance.
(adsorbed) BRP or the Intemational Standard for diphtheria Peroxidase-conjugated rabbit or goat antibody directed
toxoid (adsorbed). against guinea-pig-IgG is added, followed by a peroxidase
substrate. Optical density is measured and the relative buffer. Add 100 ~lL of peroxidase substrate to each well.
antibody titre is calculated using the usual statistical methods Allow to stand at room temperature, protected from light, for
(for example, 5.3). 30 min. Read the pi ates at 405 nm in the same order as
addition of substrate was made.
Reagents and equipment
Determination of antibody titre in guinea-pig serum by
- ELISA plates: 96 wells, columns 1-12, rows A-H.
Vero cell assay The method used relies either on
- Diphtheria guinea-pig antiseru111 (for vaccines-hwnan use) metabolic inhibition (method 1) or on cytotoxicity (method
(positive control serum), obtained by irnmunisation of 2) as the end point, and on either microscopic (cell
guinea-pigs using diphthen'a vaccine (adsorbed) BRP. morphology) or visual (colour) inspection of the cells.
- Peroxidase conjugate. Peroxidase-conjugated rabbit or goat
The limit of detection is specific for each antitoxin and is
antibody directed against guinea-pig IgG. usually berween 0.015 IU/mL (method 1) and 0.05 ID/mL
- Diphtheria toxoid. (method 2).
- Carbonate coating buffer pH 9.6. Dissolve 1.59 g of The endpoint is taken as the highest serum dilution
anhydrous sodiu111 carbonate R and 2.93 g of sodium protecting cells from the diphtheria toxin effect.
hydrogen carbonate R in 1000 mL of water R. Distribute The antitoxin activity is calculated with respect to guinea-pig
into 150 mL bottles and sterilise by autoclaving at 121 oC or WHO reference standard, and expressed in Intemational
for 15 mino Units per millilitre.
- Phosphate-buffered saline pH 7.4 (PBS). Dissolve with
stirring 80.0 g of sodium chloride R, 2.0 g of potassium
- Flat-bottomed tissue culture plates: 96 wells, columns 1-12,
dihydrogen phosphate R , 14.3 g of disodium hydrogen
rows A-H.
phosphate dihydrate R and 2.0 g of potassium chloride R in
1000 mL of water R. Store at room temperature to - 75 cm 2 tissue culture fiasks.
prevent crystallisation. Dilute to 10 times its volume with - Diphtheria toxin.
water R before use. - Diphtheria guinea-pig antiserwn (for vaccines-human use)
- Citric acid solution. Dissolve 10.51 g of citric acid R in (positive control serum), obtained by immunisation of
1000 mL of water R and adjust the solution to pH 4.0 guinea-pigs with diphthen'a vaccine (adsorbed) BRP.
with a 400 gIL solution of sodium hydroxide R . - Vero cells (African Green Monkey kidney cells). Cell
- Washing buffer. PBS containing 0.5 gIL of polysorbate 20 R. passages from P2 to P15 are suitable for use.
- Dz7uent blocking buffer. PBS containing 0.5 gIL of Method 1 The diphtheria toxin causes a cytopathogenic
polysorbate 20 R and 25 giL of dried skirnmed milk. effect on Vero cells leading to cellular Iysis. Antibodies
- Peroxidase substrate. Shortly before use, dissolve 10 mg of directed against diphtheria toxin may inhibit this
diammonium 2,2 ' -azinobis(3-ethylbenzothiazoline-6- cytopathogenic effect. Consequently, the potency of a
sulfonate) R (ABTS) in 20 mL of citric acid solution. diphtheria vaccine may be indirectly determined with the
Immediately before use add 5 ¡.¡L of strong hydrogen help of this cell culture system if different serum dilutions
peroxide solution R. from irnmunised animals are cultured with a constant toxin
concentration. In the Vero cell assay, yellow colour indicates
Method The description below is given as an example of a
viable cells, red colour dead cells. When only pan of the
suitable plate layout but others may be used. Wells lA-H are
cells are dead, the colour may be orange.
for negative control serum and wells 2A-H and 12A-H are
for positive control serum for assay monitoring. Wells 3-11A- Reagents and equipment
H are for test samples. - Modified MEM. Minimum Essential Medium (MEM)
Coat each well of the EUSA plates with 100 ¡.¡L of with Earle's Salts, without L-glutamine and sodium
diphtheria toxoid solution (0.5 Lfi'mL in carbonate coating bicarbonate.
buffer pH 9.6). Allow to stand ovemight at 4 oC in a humid - Modified medium 199. Medium 199, with Hanks' Solution
atrnosphere. To avoid temperature gradient effects, do not and L-glutarnine, without sodium bicarbonate.
stack more than 4 plates high. On the following day, wash - Poetal bovine serum.
the plates thoroughly with washing buffer. Block the plates
- Sodium bicarbonate 7.5 per cent solution.
by addition of 100 ¡.¡L of diluent block buffer to each well.
Incubate in a humid atrnosphere at 37 oC for 1 h. Wash the - Trypsin solution: trypsin 2.5 per cent solution.
plates thoroughly with washing buffer. Place 100 ¡.¡L of - EDTA solution: EDTA 0.02 per cent (Versene 1:5000)
diluent block buffer in each well of the plates, except those of solution.
row A. Prepare suitable dilutions of negative control serum, - Modified D-PBS. Dulbecco's phosphate buffered saline
positive control serum (from about 0.01 IU/mL) and test (D-PBS), without calcium, or magnesium.
sera. Allocate the negative control serum to column 1, - L-glutamine 200mM solution.
positive control serum to columns 2 and 12 and test sera to
- Penicillin/streptomycin solution.
columns 3-11 and add 100 ¡.¡L of each serum to the first
2 wells of the column to which it is allocated. Using a - Pn'mary culture medium. To 50 mL of modified MEM add
multichannel micropipette, make twofold serial dilutions from 440 mL of water R, 5 mL of L-glutamine 200 mM
row B, down the plate to row H, by transferring 100 ¡.tL solution, and 10 mL of sodium bicarbonate 7.5 per cent
from one well to the next well. Discard 100 ¡.tL from the last solution. To 25 mL of this medium add 1.25 mL of foetal
row so that all wells contain 100 ~tL. Incubate at 37 oC for bovine serum.
2 h . Wash thoroughly with washing buffer. Prepare a suitable - Maintenance culture medium. Similar to the primary culture
dilution (a 2000-fold dilution has been found to be suitable) medium except that 0.5 mL instead of 1.25 mL of foetal
of peroxidase conjugate in diluent block buffer and add bovine serum is added to 20 mL of the enriched MEM
100 ¡.¡L to each well. Incubate at 37 oC in a humid medium.
atrnosphere for I h. Wash the plates thoroughly with washing
2014 Appendix XIV K V-A423
- Medium A. To 50.0 mL of modified medium 199 add For ca1culations of potency, it must be remembered that the
440.0 mL of water R, 5.0 mL of L-glutamine 200 mM endpoint may be between a negative well and a
solution and 10.0 mL of sodium bicarbonate 7.5 per cent positive/negative wel!.
solution. Method 2 Thiazolyl blue MTT is reduced to a bluelblack
- Medium E. To 150.0 mL of medium A add 3.0 mL of formazan product by the mitochondrial dehydrogenase of
foetal bovine serum and 0.3 mL of penicillinlstreptomycin viable cells, and thus serves as a quantitative measure of
solution. living celIs present, indicating when the toxin has been
- M edium C. To 22.0 mL of medium A add 0.44 mL of neutralised by the antitoxin. White or colourless weHs
foetal bovine serum and 0.44 mL of indicate absence of viable celIs due to insufficient antitoxin
penicillinlstreptomycin solution. to neutralise the toxin.
Vero cells are cultured in tissue culture fiasks (for example Reagents and equipment
75 cm 2 /250 mL) in an incubator at 36 ± 1 °C, 5 per cent - MEM (Minimal Essential Media).
CO 2 and 90 per cent relative humidity. Vero cells are first - Newborn calf serUln.
grown in the primary culture medium. After 2-3 days of
- Antibiotic solution (containing 10 000 units of penicillin,
growth, the primary culture medium is replaced by the
10 mg of streptomycin and 25 ~lg of amphotericin B per
maintenance culture medium. When a confiuent monolayer
millilitre) .
is obtained, the culture supematant is discarded and the cell
layer washed gently with modified D-PBS. Add a mixture of - L-glutamine 200mM solution.
1 volume of trypsin solution and 1 volume of EDTA solution - Trypsin-EDTA .
to the fiask. Swirl the fiask gently and incubate in the CO 2 - Thiazolyl blue MTT [3-( 4,5-dimethylthiazol-2-yl)-2,5-
incubator for about 3 min until the cells start to break from diphenyltetrazolium bromide l.
the monolayer. Vigorously tap the side of the fiask to make - 1 M HEPES buffer pH 8.1. Dissolve 18.75 g of HEPES in
the cells fal!. Resuspend the cells in 5-6 mL of fresh medium 82 .5 mL of water R and 30.0 mL of 2 M sodium
C to obtain a homogeneous suspension. Prepare a cell hydroxide R.
suspension in medium C containing approximately 1 x
- Glucose solution (10 per cent).
10 5 cells/mL.
- Complete culture medium. Mix 200 mL of MEM with
Place 25 ~IL of medium B in each well except those of
10 mL ofnewbom calfserum, 3.0 mL of 1 M HEPES
column l. Place 25 ~lL of the diphtheria guinea-pig
buffer pH 8.1, 2.0 mL of glucose solution (10 per cent),
antiserum (for yac cines-human use) (positive control serum,
2.0 mL of antibiotic solution and 2.0 mL of I-glutamine
working dilution in medium B of 0.40 IU/mL) in wells Al,
200mM solution.
A2 and Al!. Place 25 ~IL of guinea-pig serum samples in
wells B-G of columns 1, 2 and 11. Place 25 I1L of negative - Phosphate-buffered saline pH 7.4 (PES). Dissolve 10.0 g of
control serum in row H of columns 1,2 and 11. Using a sodiUln ehloride R, 0.75 g of potassium chloride R, 1.44 g of
multichannel micropipette, make twofold serial dilutions disodium hydrogen phosphate R, and 0.125 g of potassium
across the plate (from column 2 up to column 10 for rows dihydrogen phosphate R in water R, and dilute to
A-G and up to column 8 for row H). Discard 25 ~lL from 1000.0 mL with the same solvent. Adjust the pH if
the wells in column 10 in rows A-G, and from well H8. necessary. Autoclave at 120 oC for 15 mino
Reconstitute the diphtheria toxin with saline solution to give - Thiazolyl blue MTT solution. Dissolve 0.1 g of thiazolyl
a solution of 50 IU/mL. Prepare a 50-fold dilution of this blue MTT in 20 mL of PBS . Sterilise by filtration
diphtheria toxin dilution in medium B to obtain a working (0.2 11m) and store in dark bottle.
solution of 1.0 IU/mL. Add 25 pL of this working solution to - pH adjuster solution. Mix 40 mL of acetic acid R with
wells A12 and B12 (toxin control). Make twofold serial 1.25 mL of 1 M hydrochlO1ic acid and 8.75 mL of water R.
dilutions by tranferring 25 pL from one well to the next, - Extraction buffer pH 4.7. Dissolve 10 g of sodium
from well B12 down to H12. Change the tip between each laurilsulfate R in water R and add 50 mL of
dilution. Discard 25 pL from well H12. Add 25 !1L of dimethyiformamide R, and dilute to 100 mL with water R.
medium B to wells B12-HI2. Then, place 25 ~lL ofthe Adjust the pH with an appropriate volume of pH adjuster
working dilution of the diphtheria toxin (1.0 IU/mL) in each solution.
well ofrows A-H, from column 1-10, except in wells H9 and Vero cells are cultured in tissue culture fiasks (for example
H10 (cells only, without serum and without toxin). 75 cm 2/250 mL) in an incubator at 36 ± 1 °C, 5 per cent
Cover the plates with lids or sealer and shake gently. CO 2 and 90 per cent relative humidity. Vero cells are grown
a
Incubate the plates for at least 2 h in humid container in a in the complete culture medium. After 6-7 days of growth, a
CO 2 incubator at 37 oc. Add 200 ~IL of cell suspension confiuent monolayer is obtained, the culture supematant is
containing 1 x 10 5 cells/mL to aH the wells. Cover the plates discarded and' the ceH layer is washed 3 times with trypsin-
with sealer. Incubate at 37 oC for 5 days. Check for EDTA: gently pipette out the medium, add 0.5-1 mL of
microbial contamination by microscopic examination. trypsin-EDTA, swirl the fiask and tip the trypsin out. Do this
Yellow wells are recorded as negative and red weHs indicate twice, and the 3 rd time, place the fiask in the incubator for
dead ceHs and are recorded as positive . A colour between 5 min until the celIs start to break from the monolayer.
yeHow and red indica tes a mixture of viable and dead cells Vigorously tap the side of the fiask to make the cells fal!.
and is recorded as positive/negative. The results based on the Resuspend the celIs in 6-25 mL of fresh complete culture
change in colour can be confirmed by reading viable and medium to obtain a homogeneous suspension. Prepare a ceH
dead cells under the microscope. suspension in complete culture medium containing
The potency of the guinea-pig antiserum samples is obtained approximately 4 x lOS cells/mL.
by comparing the last well of the standard preparation Place 50 ~lL of complete culture medium in each well except
showing complete neutralisation of the toxin, with the last those of column l. Place 100 I1L of diphtheria guinea pig
well of the sample demonstrating the same effect. antiserum (for vaccines-human use) (positive control serum,
V -A424 Appendix XIV K 2014
working dilution in complete culture medium of 0.12 Distribute the mice in 6 groups of not fewer than 16 and
IU/mL) in well Al and 50 llL in well A1l. Place 100 ¡.¡L of 4 groups of 10. The mice must all be of the same sex or the
guinea pig test serum samples, diluted if necessary, in wells males and females distributed equally between the groups.
B 1-G l. Add 50 llL of the same sample to wells B 11-G 11 in Selection of the challenge strain and preparation of the
the corresponding row. Place 100 llL of negative control chal1enge suspension Select a suitable strain of B.
serum in well H1 and 50 llL in well Hll. Using a multi- pertussis capable of causing the death of mice within 14 days
channel micropipette, make twofold serial dilutions by of intracerebral injection. If more than 20 per cent of the
transferring 50 llL from one well to the next working across mice die within 48 h of the injection the strain is not
the plate (from column 1-10 for rows A-G and from column suitable. Make one sub culture from the strain and suspend
1-8 for row H). the harvested B. pertussis in a solution containing 10 giL of
Diphtheria toxin of known activity and Lf content is diluted easein hydrolysate R and 6 giL of sodium eh/oride R and having
to a suitable working stock containing at least 4 minimum a pH of 7.0 to 7.2 or in another suitable solution. Determine
cytopathic doses in complete culture medium. Add 50 llL of the opacity of the suspension. Prepare a series of dilutions in
the diluted toxin to each well except H9 and Hlü (cell the same solution and allocate each dilution to a group of
control), A11-H11 (serum control) and A12-H12 (toxin 10 mice. Inject intracerebrally into each mouse adose
contro!). Add 100 llL of diluted toxin to well A12 and make (0.02 mL or 0.03 mL) of the dilution allocated to its group.
twofold serial dilutions by transferring 50 llL from one well After 14 days, count the number of mice surviving in each
to the next working down the plate (from well A12-H12). group. From the results, calculate the expected opacity of a
Discard 50 llL from well H12. Add 50 ~lL of complete suspension containing 100 LDso in each challenge dose.
medium to wells H9 and HI0 . For the test of the vaccine to be examined make a fresh
Cover the plates with a lid or sealer and leave for 1 h at sub culture from the same strain of B. pertussis and prepare a
room temperature to allow toxin neutralisation to occur. suspension of the harvested organisms with an opacity
50 llL of cell suspension containing approximately 4 x corresponding to about 100 LDso in each challenge dose.
105 cells/mL is added to each well. The plates are sealed and Prepare 3 dilutions of the challenge suspension.
incubated at 37 oC for 6 days. Check for microbial Determination of potency Prepare 3 serial dilutions of
contamination by microscopic examination. 10 llL of the vaccine to be examined and 3 similar dilutions of the
thyazolyl blue MTT solution is added to each well. reference preparation such that in each the intermediate
T.he plates are incubated at 37 oC for a further 2-4 h. Then, dilution may be expected to protect about 50 per cent of the
the medium is removed and 100 ).lL of extraction buffer mice fro!TI the lethal effects of the challenge dose of B.
pH 4.7 is added to each well. The plates are incubated at pertussis. Suggested doses are 1/8, 1/40 and 1/200 of the
37 oC and left overnight to aid the extraction process. Once human dose of the vaccine to' be examined and 0.5 IU,
extraction and solubilisation is complete, plates are visually 0.1 IU and 0.02 IU of the reference preparation, each dos e
examined or read at 570 nm. being contained in a volume not exceeding 0.5 mL. Allocate
Blue/black wells are recorded as negative (all the cells are the 6 dilutions, one to each of the groups of not fewer than
alive, toxin neutralisation by antitoxin) and white or 16 mice, and inject intraperitoneally into each mouse one
colourl'ess wells indicate dead cells (no toxin neutralisation) dose of the dilution allocated to its group. After 14 - 17 days
and are recorded as positive. inject intracerebrally into each animal in the groups of not
The potency of the test antitoxin is obtained by comparing fewer than 16, one dose of the challenge suspension.
the last well of the reference antitoxin preparation showing Allocate the challenge suspension and the 3 dilutions made
neutralisation of the toxin, with the last well of the antitoxin from it, one to each of the groups of 10 mice, and inject
preparation demonstrating the same effect. The neutralising intracerebrally one dose of each suspension into each mouse
antibody titre of the sample being examined can be in the group to which that suspension is allocated. Exclude
calculated by multiplication of the dilution factor with total from consideration any mice that die within 48 h of
number of International Units per millilitre of the reference challenge. Count the number of micesurviving in each of
preparation at the end point. The test is valid if all the cells the groups after 14 days. Calculate the potency of the
in the toxin control are dead and reference antitoxin gives a vaccine to be examined relative to the potency of the
neutralisation in at least the first 2 dilutions tested. reference preparation on the basis of the numbers of animals
surviving in each of the groups of not fewer than 16.
2. Assay ofpertussis vaccine (Whole Cell) The test is not valid unless:
(Ph. Eur. method 2.7.7) - for both the vaccine to be examined and the reference
The potency of pertussis vaccine (whole cel!) is determined preparation, the 50 per cent protective dose lies between
by comparing the dose necessary to protect mice against the the largest and the smallest doses given to the rnice;
effects of a lethal dose of Bordetella pertussis, administered - the flumber of animals that die in the 4 groups of
intracerebrally, with the quantity of a reference preparation, 10 injected with the challenge suspension and its dilutions
calibrated in International Units, needed to give the same indicates that the challenge dose is approximately
protection. 100 LDso; and
The International Unit is the activity contained in a stated - the statistical analysis shows no deviation from linearity or
amount of the International Standard which consists of a parallelism.
quantity of dried pertussis vaccine. The equivalence in The test may be repeated but when more than one test is
Intemational Units of the International Standard is stated by performed the results of all val id tests must be combined.
the World Health Organization.
Selection and distribution ofthe test animals Use in 3. Assay of tetanus vaccine (adsorbed)
the test healthy mice les s than 5 weeks old of a suitable (Ph. Eur method 2.7.8)
strain from the same stock, the difference in mass between The potency of tetanus vaccine is determined by
the heaviest and the lightest being not greater than 5 g. administration of the vaccine to animals (guinea-pigs or
2014 Appendix XIV K V -A425
mice) foJIowed either by challenge with tetanus toxin Where a single-dilution assay is used, production and test
(method A or B) or by determination of the titre of consistency over time are monitored via suitable indicators
antibodies against tetanus toxoid in the serum of the guinea- and by carrying out a full multiple-dilution assay periodically,
pigs (method C). In both cases, the potency of the vaccine is for example every 2 years. For serological assays, suitable
calculated by comparison with a reference vaccine, calibrated indicators to monitor test consistency are:
in lnternational Units. For methods A and B, in countries where - the mean and standard deviation of relative antitoxin titres
the paralysis method is not obligarory, the LDso method may be or scores of the serum samples obtained after
used. For the LDso method, the number of animals and the administration of a fixed dose of the vaccine reference
pl'Ocedure are identical ro those described for the pamlysis method, preparation;
but the end-point is the death of the animalmther ¡han paralysis.
- the antitoxin titres or scores of run controls (positive and
The International Unit is the activity contained in a stated negative serum samples);
amount of the International Standard for tetanus toxoid - the ratio of antitoxin ti tres or scores for the positive serum
(adsorbed). The equivalen ce in International Units of the control to the serum samples corresponding to the
International Standard is stated by the World Health reference vaccine.
Organisation.
Method A. Challenge test in guinea pigs
Tetanus vaccine (adsorbed) BRP is calibrated in International
Units with reference to the International Standard. Selection and distribution ofthe test animals Use in
the test healthy guinea-pigs from the same stock, each
The method chosen for the assay of tetanus vaccine
weighing 250-350 g. Use guinea-pigs of the same sex or with
(adsorbed) depends on the intended purpose. Method A
males and females equaJIy distributed between the groups.
or Bis used:
Distribute the guinea-pigs in not fewer than 6 equal groups;
l. during development of a vaccine, to assay batches use groups containing a number of animals sufficient to
produced to validate the production; obtain results that fulfil the requirements for a valid assay
2. wherever revalidation is needed following a significant prescribed below. lf the activity of the challenge toxin has to
change in the manufacturing process. be determined, include 3 further groups of 5 guinea-pigs as
Method A or B may also be used for the routine assay of unvaccinated controls.
batches of vaccine, but in the interests of animal welfare, Selection of the challenge toxin Select a preparation of
method C is used wherever possible. tetanus toxin containing not less than 50 times the
Method C may be used, except as specified under 1 and 2 50 per cent. paralytic dose per millilitre. lf the challenge toxin
aboye, after verification of the suitability of the method for preparation has been shown to be stable, it is not necessary
the product.' For this purpose, a suitable number of batches to verify tlle paralytic dose for every assay.
(usually 3) are assayed by method C and method A or B. Preparation of the challenge toxin solution
Where different vaccines (monovalent or combinations) are Immediately before use, dilute the chaJIenge toxin with a
prepared from tetanus toxoid of the same origin and with suitable diluent (for example, peptone buffered saline
comparable levels (expressed in LflmL) of the same tetanus solution pH 7.4) to obtain a stable challenge toxin solution
toxoid, suitability demonstrated for the combination with the containing approximately 50 times the 50 per cent paralytic
highest number of components can be assumed to be valid dose per millilitre. If necessary, use portions of the challenge
for combinations with fewer components and for monovalent toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the
vaccines. Any combinations containing a whole-ceJI pertussis same diluent to determine the activity of the toxin.
component or containing haemophilus type b conjugate Dilution ofthe test and reference preparations Using a
vaccine with tetanus toxoid in the same vial must always be 9 gIL solution of sodium chloride R, prepare dilutions of the
assessed separately. vaccine to be examined and of the reference preparation,
For combinations containing diphtheria and tetanus such that for each, the dilutions form a series differing by
components, the serological assay (method C) can be not more than 2.5-fold steps and in which the intermediate
performed with the same group of animals used for the dilutions, when injected subcutaneously at adose of 1.0 mL
serological assay of the diphtheria vaccine (adsorbed)'(2. 7. 6) per guinea-pig, protect approximately 50 per cent of the
when the common immunisation conditions for the tetanus animals from the paralytic effects of the subcutaneous
and the diphtheria components (for example, doses, injection of the quantity of tetanus toxin prescribed for this
duration) have been demonstrated 'to be valid for the test.
combined vaccine. Immunisation and challenge Allocate the dilutions, 1 to
The design of the assays described below uses multiple each of the groups of guinea-pigs, and inject subcutaneously
dilutions for the test and reference preparations. Based on 1.0 mL of each dilution into each guinea-pig in the group to
the potency data obtained in multiple-dilution assays, it may which that dilution is allocated. After 28 days, inject
be possible to reduce the number of animals needed to subcutaneously into each animal 1.0 mL ofthe challenge
obtain a statisticaJIy significant result by applying a simplified toxin solution (containing 50 times the 50 per cent paralytic
model su eh as a single dilution for bQth test and reference dose) .
preparations. Such a model enables the analyst to determine Determination of the activity of the challenge toxin If
whether the potency of the test preparation is significantly necessary, allocate the 3 dilutions made from the challenge
higher than the minimum required, but does not give toxin solution, 1 to each of the 3 groups of 5 guinea-pigs,
information on the dose-response curves and their linearity, and inject subcutaneously 1.0 mL of each solution into each
parallelism and significant slope. The simplified model allows guinea-pig in the group to which that solution is allocated.
for a considerable reduction in the number of animals The activity and stability of the challenge toxin are
required and must be considered by each analyst in determined by carrying out a suitable number of
accordance with the provisions of the European Convention determinations of the 50 per cent paralytic dose . It is then
for the protection of vertebrate animals used for experimental not necessary to repeat the determination for each assay.
and other scientific purposes.
Reading and interpretation of results Examine the 0.5 mL of each dilution into each mouse in the group to
guinea-pigs twice daily. Remove and euthanise all animals which that dilution is allocated. After 28 days, inject
showing definite signs of tetanus paralysis. Count the subcutaneously into each animal 0.5 mL of the challenge
number of guinea-pigs without paralysis 5 days after injection toxin solution (containing 50 times the 50 per cent paralytic
of the challenge toxin. Calculate the potency of the vaccine dose).
to be examined relative to the potency of the reference Determination of the activity of the challenge toxin If
preparation on the basis of the proportion of challenged necessary, allocate the 3 dilutions made from the challenge
animal s without paralysis in each group of vaccinated guinea- toxin solution, 1 to each of the 3 groups of not fewer than
pigs, using the usual statistical methods (for example, 5.3). 5 mice, and inject subcutaneously 0.5 mL of each solution
Requirements for a valid assay The test is not valid into each mouse in the group to which that solution is
unless: allocated.
- for both the vaccine to be examined and the reference Reading and interpretation of results Examine the mice
preparation, the 50 per cent protective dose lies between twice daily. Remove and euthanise all animals showing
the largest and smallest doses of the preparations given to definite signs of tetanus paralysis. Count the number of mice
the guinea-pigs; without paralysis 4 days after injection of the challenge toxin.
- where applicable, the number of paralysed animals in the Calculate the potency of the vaccine to be examined relative
3 groups of 5 injected with the dilutions of the challenge to the potency of the reference preparation on the basis of
toxin solution indica tes that the challenge was the proportion of challenged animals without paralysis in
approximately 50 times the 50 per cent paralytic dose; each group of vaccinated mice, using the usual statistical
- the confidence limits (P = 0.95) are not les s than methods (for example, 5.3).
50 per cent and not more than 200 per cent of the Requirements for a valid assay The test is not valid
estimated potency; unless:
- the statistical analysis shows a significant slope and no - for both the vaccine to be examined and the reference
deviation from linearity and parallelism of the dose- preparation, the 50 per cent protective dos e lies between
response curves (chapter 5.3 describes possible alternatives the largest and smallest doses of the preparations given to
if significant deviations are observed). the mice;
The test may b¡; repeated but when more than 1 test is - where applicable, the number of paralysed ' animals in the
performed the results of all valid tests must be combined in 3 groups of not fewer than 5 injected with the dilutions of
the estimate of potency. the challenge toxin solution, indicates that the challenge
Method B. Challenge test in mice dos e was approximately 50 times the 50 per cent paralytic
Selection and distribution of the test animals Use in dose;
the test healthy mice from the same stock, about 5 weeks old - the confidence limits (P = 0.95) are not less than
and from a strain shown to be suitable. Use mice of the 50 per cent and not more than 200 per cent of the
same sex or with males and females equally distributed estimated potency;
between the groups . Distribute the mice in not fewer than 6 - the statistical analysis shows a significant slope and no
equal groups; use groups containing a number of animals deviation from linearity and parallelism of the dose-
sufficient to obtain results that fulfil the requirements for a response curves (chapter 5.3 describes possible alternatives
val id assay prescribed below. If the challenge toxin to be if significant deviations are observed).
used has not been shown to be stable or has not been The test may be repeated but when more than 1 test is
adequately standardised, include 3 further groups of not performed the results of all valid tests must be combined in
fewer than 5 mice to serve as unvaccinated controls. the estimate of potency.
Se1ection ofthe challenge toxin Select a preparation of
tetanus toxin containing not les s than 100 times the Method C. Determination 01 antibodies in guinea pigs
50 per cent paralytic dose per millilitre. If the challenge toxin Selection and distribution of the test animals U se in
preparation has been shown to be stable, it is not necessary the test healthy guinea-pigs from the same stock, each
to verify the paralytic dose for every assay. weighing 250-350 g. Use guinea-pigs of the same sex or with
Preparation of the challenge toxin solution males and females equally distributed between the groups.
Immediately befare use, dilute the challenge toxin with a Distribute the guinea-pigs in not fewer than 6 equal groups;
suitable diluent (for example, peptone buffered saline use groups containing a number of animals sufficient to
solution pH 7.4) to obtain a stable challenge toxin solution obtain results that fulfil the requirements for a valid assay
containing approximately 50 times the 50 per cent paralytic prescribed below. Use a further group of non-vaccinated
dos e in 0.5 mL. If necessary, use portions of the challenge guinea-pigs of the same origin to provide a negative serum
toxin solution diluted 1 to 16, 1 to 50 and 1 to 160 with the control. If test consistency has been demonstrated, a
same diluent to determine the activity of the toxin. reference negative serum control may be used.
Dilution of the test and reference preparations U sing a Reference preparation Use a suitable reference
9 gIL solution of sodium chloride R, prepare dilutions of the preparation such as tetanus vaccine (adsorbed) BRP or a
vaccine to be examined and of the reference preparation, batch of vaccine shown to be effective in clinical studies, or a
such that for each, the dilutions form a series differing by batch representative thereof, and which has been calibrated
not more than 2.5-fold steps and in which the intermediate in International Units with reference to tetanus vaccine
dilutions, when injected subcutaneously at adose of 0.5 mL (adsorbed) BRP or the International Standard for tetanus
per mouse, protect approximately 50 per cent of the animals toxoid (adsorbed).
from the paralytic effects of the subcutaneous injection of the Dilution of the test and reference preparations U sing a
quantity of tetanus toxin prescribed for this test. 9 gIL solution of sodium chloride R as diluent, prepare serial
Immunisation and challenge Allocate the dilutions, 1 to dilutions of the vaccine to be examined and the reference
each of the groups of mice, and inject subcutaneously preparation; series differing by 2.5- to 5-fold steps have been
found to be suitable. Use not fewer than 3 dilutions within below. The scale gives typical signs when injection of the
the range of, for example, 0.5-16 IU/mL for each series. challenge toxin is made in the dorsal region, close to one of
Use the dilutions for irnmunisation preferably within 1 h of the hind legs. Grade T3 is taken as the end-point, but with
preparation. AlIocate 1 dilution to each group of guinea-pigs. experience grade T2 can be used instead. Tetanus toxin
Irnmunisation Inject subcutaneously to each guinea-pig produces in the toxin-injected hind leg paresis followed by
1.0 mL of the dilution allocated to its group. paralysis that can be recognised at an early stage.
Blood sampling 35-42 days after immunisation, take a The tetanus grades in mice are characterised by the following
signs:
blood sample from each vaccinated and control guinea-pig
using a suitable method. - TI : slight stiffness of toxin-injected hind leg, only
observed when the mouse is lifted by the tail;
Preparation of serum samples Avoid frequent freezing
and thawing of serum samples. To avoid microbial - T2: paresis of the toxin-injected hind leg, which still can
contamination, it is preferable to carry out manipulations in function for walking;
a laminar-fiow cabinet. - T3: paralysis of the toxin-injected hind leg, which does
Determination of antibody titre Determine the relative not function for walking;
antibody titre or score of each serum sample by a suitable - T4: the toxin-injected hind leg is completely stiff with
irnmunochemical method (2.7.1). The methods shown below irnmovable toes;
(enzyme-linked immunosorbent assay (ELISA) and toxin- - T5: tetanus seizures, continuous tonic spasm of muscles;
binding inhibition (ToBI)) have been found to be suitable. - D: death.
Calculation of potency Calculate the potency of the Method C. Determination of antibodies in guinea pigs
vaccine to be examined in Intemational Units relative to the
Preparation of serum samples For the preparation of
reference preparation, using the usual statistical methods (for
serum samples, the following technique has been found to be
example, 5.3).
suitable. Inven the tubes· containing blood samples 6 times
Requirements for a valid assay The test is not valid and allow to stand at 37 oC for 2 h, then at 4 oC for 2 h.
unless: Centrifuge at room temperature at 800 g for 20 min.
- the confid~nce limits (P = 0.95) are not les s than Transfer the serum to sterile tubes and store at a
50 per cent and not more than 200 per cent of the temperature below - 20 ce. At least a 40 per cent yield of
estimated potency; serum is obtained by this procedure.
- the statistical analysis shows a significant slope and no Determination of antibody titre The ELISA and ToBI
deviation from linearity and parallelism of the dose- tests shown below are given as examples of immunochemical
response curves (chapter 5.3 describes possible altematives methods that have been found to be suitable for the
if significant deviations are observed). determination of antibody titre.
The test may be repeated but when more than 1 test is Determination of antibody titre in guinea-pig serum by
performed the results of all valid tests must be combined in enzyme-linked im.munosorbent assay (ELISA)
the estimate of potency. Dilutions of test and reference sera are made on ELISA
The following section is published for information. plates coated with tetanus toxoid. A positive guinea-pig
serum control and a negative guinea-pig serum control are
Assay of tetanus vaccine (adsorbed): guidelines included on each plate to monitor the assay performance.
Method A. Challenge test in guinea pigs Reading and Peroxidase-conjugated rabbit or goat antibody directed
interpretation of resuIts. In order to minimise suffering in the against guinea-pig-IgG is added, followed by a peroxidase
test animals, it is recommended to note the degree of substrate. Optical density is measured and the relative
paralysis on a scale such as that shown below. The scale antibody titre is calculated using the usual statistical methods
gives typical signs when subcutaneous injection of the (for example, 5.3).
challenge toxin is made mid-ventrally, directly behind the Reagents and equipment
stemum with the needle pointing towards the neck of the - ELISA plates: 96 wells, columns 1-12, rows A-H.
guinea-pig. Grade T3 is taken as the end-point, but with - Clostridium tetani guinea-pig antisernm (for vaccines-human
experience grade T2 can be used instead. Tetanus toxin use) BRP (positive control serum).
produces in at least 1 of the forelimbs paralysis that can be - Peroxidase conjugate. Peroxidase-conjugated rabbit or goat
recognised at an early stage. The tetanus grades in guinea- antibody directed against guinea-pig IgG.
pigs are characterised by the following signs:
- Tetanus toxoid.
- TI: slight stiffness of 1 forelimb, but difficult to observe; - Carbonate coating buffer pH 9.6. Dissolve 1.59 g of
- T2: paresis of 1 forelimb which still can function; anhydrous sodium carbonate R and 2.93 g of sodium
- T3: paralysis of 1 forelimb . The animal moves reluctantly, hydrogen carbonate R in 1000 mL of water R. Distribute
the body is often slightly banana-shaped owing to into 150 mL bottles and sterilise by autoclaving at 121 °C
scoliosis; for 15 min o
- T4: the forelimb is completely stiff and the toes are - Phosphate-buffered saline pH 7.4 (PES). Dissolve with
immovable. The muscular contraction of the forelimb is stirring 80.0 g of sodium ehloride R, 2.0 g of potassium
very pronounced and usually scoliosis is observed; dihy dmgenphosphate R, 14.3 g of disodium hydrogen
- T5: tetanus seizures, continuous tonic spasm of muscles; phosphate dihydrate R and 2.0 g of potassium ehloride R in
1000 mL of water R . Store at room temperature to
- D: death.
prevent crystallisation. DiJute to 10 times its volume with
Method B. Challenge test in mice water R befo re use.
Reading and interpretation of results In order to - Citrie acid solution. Dissolve 10.51 g of eitrie acid R in
minimise suffering in the test animals, it is recommended to 1000 mL of water R and adjust the solution to pH 4.0
note the degree of paralysis on a scale such as that shown with a 400 gIL solution of sodium hydrcxide R.
- Washing buffer. PBS containing 0.5 gIL of polysorbate 20 R . and 0.2 g of sodium azide R in 1000 mL of water R, adjust
- Diluent block buffer. PBS containing 0.5 giL of polysorbate to pH 9.6 and autoclave at 121 °C for 20 mino
20 R and 25 gIL of dried skimmed milk. - Sodium aceta te buffer pH 5.5. Dissolve 90 .2 g of anhydrous
- Peroxidase substrate. Shortly before use, dissolve 10 mg of sodium acetate R in 900 mL of water R, adjust to pH 5.5
diammonium 2,2' -azinobis (3-ethylbenzothiazoline-6-sulfonate) using a saturated solution of citric acid monohydrate R and
R (ABTS) in 20 mL of citric acid solution. Immediately dilute to 1000 mL with water R.
before use add 5 )lL of strong hydrogen peroxide solution R. - Phosphate-buffered saline pH 7.2 (PBS). Dissolve 135.0 g of
Method The description below is given as an example of a sodium chloride R, 20.55 g of disodium hydrogen phosphate
suitable plate layout but others may be used. Wells lA-H are dihydrate R and 4.80 g of sodium dihydrogen phosphate
for negative control serum and wells 2A-H and 12A-H are monohydrate R in water R and dilute to 15 L with the
for positive control serum for assay monitoring. Wells 3-11A- same solvent. Autoclave at 100 oC for 60 mino
H are for test samples. - Diluent buffer. PBS containing 5 gIL of bovine albumin R
Coat each well of the EUSA plates with 100 )lL of tetanus and 0.5 gIL of polysorbate 80 R.
toxoid solution (0.5 LllmL in carbonate coating buffer - Block buffer. PBS containing 5 giL of bovine albumin R.
pH 9.6) . Allow to stand ovemight at 4 oC in a humid - Tetramethylbenzidine solution. 6 gIL solution of
atrnosphere. To avoid temperature gradient effects, do not tetramethylbenzidine R in ethanol (96 per cent) R.
stack more than 4 plates high. On the following day, wash The substance dissolves within 30-40 min at room
the plates thoroughly with washing buffer. Block the plates temperature.
by addition of 100 )lL of diluent block buffer to each well. - Peroxidase substrate. Mix 90 mL of water R, 10 mL of
Incubate in a humid atrnosphere at 37 oC for 1 h. Wash the
sodium acetate buffer pH 5.5, 1.67 mL of
plates thoroughly with washing buffer. Place 100 )lL of
tetramethylbenzidine solution and 20 )lL of strong hydrogen
diluent block buffer in each well of the plates, except those of peroxide solution R.
row A. Prepare suitable dilutions of negative control sérum,
positive control serum (from about 0.01 IU/mL) and test - Washing solution. Tap water containing 0.5 giL of
sera. Allocate the negative control serum to column 1, polysorbate 80 R.
positive control serum to columns 2 and 12 and test sera to Method Block the microtitre plates by placing in each well
columns 3-11 and add 100 )lL of each serum to the first 150 )lL of block buffer. Cover the plates with a lid or sealer.
2 wells of the column to which it is allocated. Using a Incubate in a humid atrnosphere at 37 oC for 1 h. Wash the
multichannel micropipette, make twofold serial dilutions from plates thoroughly with washing solution. Place 100 )lL of
row B down the plate to row H, by transferring 100 )lL from PBS in each well. Place 100 )lL of reference guinea-pig
one well to the next. Discard 100 )lL from the last row so tetanus antitoxin in the first well of a row. Place 100 )lL of
that all wells contain 100 )lL. Incubate at 37 oC for 2 h. undiluted test sera in tbe first well of the required number of
Wash thoroughly with washing buffer. Prepare a suitable rows. Using a multichannel micropipette, make twofold serial
dilution (a 2000-fold dilution has been found to be suitable) dilutions across the plate (up to column 10), by transferring
of peroxidase conjugate in diluent block buffer and add 100 )lL from one well to the next. Discard 100 )lL from the
100 )lL to each well. Incubate at 37 oC in a humid last column so that all wells contain 100 )lL. Prepare a 0.1
atrnosphere for 1 h. Wash the plates thoroughly with washing LllmL solution of tetanus toxin or toxoid using PBS as
buffer. Add 100 )lL of peroxidase substrate to each well. diluent. Add 40 ~lL of this solution to each well except those
Allow to stand at room temperature, protected from Iight, for of column 12. The wells of column 11 are a positive control.
30 mino Read the plates at 405 nm in the same order as Add 40 )lL of PBS to the wells of column 12 (negative
addition of substrate was made. control). Shake the plates gently and cover them with lids.
DetermÍnation of antibody titre in guinea-pig serum by Coat the EUSA plates: immediately before use make a
toxin- or toxoid-binding inhibirlon (ToBI) Tetanus suitable dilution of equine anti-tetanus IgG in carbonate
toxin or toxoid is added to serial dilutions of test and buffer pH 9.6 and add 100 )lL to each well. Incubate the 2
reference sera; the serum/antigen mixtures are incubated series of plates ovemight in a humid atrnosphere at 37 oc.
ovemight. To determine unbound toxin or toxoid, the To avoid temperature gradient effects, do not stack more
mixtures are transferred to an EUSA plate coated with than 4 plates high. Cover the plates with Iids. On the
tetanus antitoxin. Peroxidase-conjugated equine anti-tetanus following day, wash the EUSA plates thoroughly with
IgG is ádded followed by a peroxidase substrate. Optical washing solution. Block the plates by placing in each well
density is measured and the antibody titre is calculated using 125 )lL of block buffer. Incubate at 37 °C in a humid
the usual statistical methods (for example, 5.3). A positive atrnosphere for 1 h . Wash the plates thoroughly with
control serum and a negative control serum are included on washing solution. Transfer 100 )lL of the pre-incubation
each plate to monitor assay performance. mixture from the polystyrene plates to the corresponding
wells of the EUSA plates, starting with column 12 and then
continuing from 1 to 11. Cover the plates with a lid.
- Round-bottomed, rigid polystyrene microtitre plates. Incubate at 37 oC in a humid atrnosphere for 2 h. Wash the
- Flat-bottomed ELISA plates. EUSA plates thoroughly with washing solution. Make a
- Tetanus toxin or tetanus toxoid. suitable dilution (a 4000-fold dilution has been found to be
- Clostridium tetani guinea-pig antiserum (for vaccines-human suitable) of the peroxidase-conjugated equine anti-tetanus
use) BRP (positive control serum). IgG in diluent buffer. Add 100 )lL of the dilution to each
well and cover the plates with a lid. Incubate at 37 oC in a
- Equine anti-tetanus IgG.
humid atrnosphere for 1.5 h. Wash the EUSA plates
- Peroxidase-conjugated equine anti-tetanus IgG. thoroughly with washing solution. Add 100 )lL of peroxidase
- Carbonate buffer pH 9.6. Dissolve 1.5 g of anhydrous substrate to each well. A blue colour deveIops. Incubate the
sodium carbonate R, 2.39 g of sodium hydrogen carbonate R pIates at room temperature. Stop the reaction at a given time
(within 10 min) by the addition of 100 ~IL of 2 M sulfuric
acid to each well in the same order as the addition of reference preparation by the competent authority in the light
substrate. The colour changes from blue to yellow. Measure of the validation data.
the absorbance at 450 nm immediately after addition of the Hepatitis A vaeeine (inaetivated, non-adsorbed) BRP is suitable
sulfuric acid or maintain the plates in the dark until reading. for use as a reference preparation.
4. Assay of hepatitis A vaccine
5. Assay of hepatitis B vaccine (rDNA)
(Ph Eur method 2. 7.14)
(Ph. Eur. method 2. 7.15)
The assay of hepatitis A vaccine is carried out either in vivo,
The assay of hepatitis B vaccine (rDNA) is carried out either
by comparing in given conditions its capacity to induce
in vivo, by comparing in given conditions its capacity to
specific antibodies in mice with the same capacity of a
induce specific antibodies against hepatitis B surface antigen
reference preparation, or in vitra, by an immunochemical
(HBsAg) in mice or guinea-pigs with the same capacity of a
determination of antigen contento
reference preparation, or in vitro, by an immunochemical
In vivo assay determination of the antigen contento
The test in mice shown below is given as an example of a In vivo assay
method that has been found suitable for a given vaccine; Selection and distribution ofthe test animals U se in
other validated methods may also be used. the test healthy mice from the same stock, about 5 weeks
Selection and distribution of the test animals Use in old. The strain of mice used for this test must give a
the test healthy mice from the same stock, about 5 weeks old significant slope for the dos e-response curve to the antigen;
and from a strain shown to be suitable. Use animals of the mice with haplotype H-2 q or H_2 d are suitable. Healthy
same sexo Distribute the animals in at least 7 equal groups of guinea-pigs weighing 300 g to 350 g (about 7 weeks old)
a number suitable for the requirements of the assay. from the same stock are also suitable. Use animals of the
Determination of potency of the vaccine to be same sexo Distribute the animals in at least 7 equal groups of
exarnined Using a 9 giL solution of sodium eh/oride R a number appropriate to the requirements of the assay.
containing the aluminium adjuvant used for the vaccine, Determination of potency of the vaccine to be
prepare at least 3 dilutions of the vaccine to be examined exarnined Using a 9 gIL solution of sodium eh/oride R
and matching dilutions of the reference preparation. AIIocate containing th¡; aluminium adjuvant used for the vaccine or
the dilutions one to each of the groups of animals and inject another appropriate diluent, prepare at least 3 dilutions of
subcutaneously not more than 1.0 mL of each dilution into the vaccine to be examined and matching dilutions of the
each animal in the group to which that dilution is aIlocated. reference preparation. AlIocate the dilutions, 1 to each of the
Maintain a group of unvaccinated controls, injected groups of animals, and inject intraperitoneally not more than
subcutaneously with the same volume of diluent. After 28 to 1.0 mL of each dilution into each animal in the group to
32 days, anaesthetise and bleed aIl animals, keeping the which that dilution is alIocated. One group of animals
individual sera separate. Assay the individual sera for specific remains unvaccinated and is injected intraperitoneally with
antibodies against hepatitis A virus by a suitable the same volume of diluent. After an appropriate time
immunochemical method (2.7.1). interval (for example, 4-6 weeks), anaesthetise and bleed the
Calculations Carry out the caIculations by the usual animals, keeping the individual sera separa te. Assay the
statistical methods for an assay with a quantal response (5.3) . individual sera for specific antibodies against HBsAg by a
From the distribution of reaction levels measured on aIl the suitable immunochemical method (2.7.1).
sera in the unvaccinated group, determine the maximum Calculations CaIculations are carried out by the usual
reaction level that can be expected to occur in an , statistical methods for an assay with a quantal response
unvaccinated animal for that particular assay. Any response (5.3).
in vaccinated animals that exceeds this level is by definition a From the distribution of reaction levels measured on aIl the
seroconversion. sera in the unvaccinated group, the maximum reaction level
Make a suitable transformation of the percentage of animals that can be expected to occur in an unvaccinated animal for
showing seroconversion in each group (for example, a pro bit that particular assay is determined. Any response in
transformation) and analyse the data according to a parallel- vaccinated animals that exceeds this level is by definition a
line log dose-response modeI. Determine the potency of the seroconversion.
test preparation relative to the reference preparation.
Make a suitable transformation of the percentage of animals
Validity conditions The test is not valid unless: showing seroconversion in each group (for example, a pro bit
- for both the test and the reference vaccine, the EDso lies transformation) and analyse the data according to a parallel-
between the' smaIlest and the largest doses given to the line log dos e-response modeI. Determine the potency of the
animals; test prepararion relative to the reference preparation.
- the statistical analysis shows no significant deviation from Validity conditions The test is not valid unless:
Iinearity or paraIlelism; - for both the test and the reference vaccine, the EDso lies
- the confidence limits (P = 0.95) are not less than between the smaIlest and the largest doses given to the
33 per cent and not more than 300 per cent of the animals;
estimated potency. - the statistical analysis shows no significant deviation from
Potency requirement The upper confidence limit Iinearity or parallelism;
(P = 0.95) of the estimated relative potency is not less than
- the confidence limits (P = 0.95) are not les s than
1.0. 33 per cent and not more than 300 per cent of the
In vitro assay estimated potency.
Carry out an irnmunochemical determination (2.7.1) of Potency requirement The upper confidence limit
antigen content with acceptance criteria validated against the (P = 0.95) of the estimated relative potency is not less than
in vivo test. The acceptance criteria are approved for a given 1.0.
In vitro assay METHOD A. SEROLOGY IN MICE

Carry out an immunochemical determination (2.7. 1) of Reference vaccine A batch of vaccine shown to be
antigen content with acceptance criteria validated against the effective in clinical trials or a batch representative thereof is
in vivo test. used as a reference vaccine. F or the preparation of a
Enzyme-linked irnmunosorbent assay (ELISA) and radio- representative batch, strict adherence to the production
irnmunoassay (RIA) using monoclonal antibodies specific for process used for the batch tested in clinical trials is
protection-inducing epi topes of HBsAg have been shown to necessary. The stability of the reference vaccine shall be
be suitable. Suitable numbers of dilutions of the vaccine to monitored and documented.
be examined and the reference preparation are used and a Reference antiserum A reference antiserum of assigned
parallel-line model is used to analyse the data, which may be activity is used in the test and serves as the basis for
suitably transformed. Kits for measuring HBsAg in vitro are ca1culation of the antibody levels in the test sera. Bordetella
commercially available and it is possible to adapt their test pertussis mouse antiserum BRP is suitable for use as a
procedures for use as an in vitro potency assay. reference antiserum.
The acceptance criteria are approved for a given reference The following test model is given as an example of a method that
preparation by the competent authority in light of the has been found lO be satisfactory.
validation data. Selection and distribution of the test animals U se in
the test healthy mice (for example, CDI strain) of the same
6. Assay of pertussis vaccine (acellular) stock, about 5 weeks old. Distribute the animals in 6 groups
(Ph. Eur. method 2.7.16) of a number appropriate to the requirements of the assay.
The capacity of the vaccine to induce the formation of Use 3 dilutions of the vaccine to be examined and 3
specific antibodies in mice or guinea-pigs is compared with dilutions of a reference preparation and attribute each
the same capacity of a reference preparation examined in dilution to a group of mice. Inject intraperitoneally or
parallel; antibodies are determined using a suitable subcutaneously into each mouse 0.5 mL of the dilution
immunochemical method (2.7.1) such as enzyme-linked attributed to its group. During validation studies a further
immunosorbent assay (ELISA) . group of mice may be used as a negative control by injecting
For combinations containing pertussis components together the animals with diluent alone.
with diphtheria and tetanus components, the serological assay Collection of serum samples 4-5 weeks after vaccination,
in guinea-pigs can be performed with the same group of bleed the mi ce individually under anaesthesia. Store the sera
animals used for the serological assay of diphtheria 'vaccine at - 20 oC until used for antibody determination.
(adsorbed) (2.7.6) and of tetanus vaccine (adsorbed) (2.7.8) Antibody determination Assay the individual sera for
when the common immunisation conditions for all content of specific antibodies to each acellular pertussis
components (for example, doses, duration) have been antigen using a validateci method such as the ELISA test
demonstrated to be valid for the combined vaccine. shown below.
The guinea-pig model allows for a further reduction in the
ELISA test Microtitre plates (poly(vinyl chloride) or
number of animals required and must be considered by each
polystyrene as appropriate for the specific antigen) are coated
analyst in accordance with the provisions of the Eutopean
with the purified antigen at a concentration of 100 ng per
Convention for the protection of vertebrate animals used for
well. After washing, unreacted sites are blocked by
experimental and other scientific purposes.
incubating the plates with a solution of bovine serum
The design of the assays A and B described below uses albumin and then washed. 2-fold dilutions of sera from
multiple dilutions for the test and reference preparations. individual mice immunised with test or reference vaccines
After validation for a given vaccine, it is possible to apply a are made on the plates. Reference antiserum is included on
simplified model such as a single dilution for both test and each plate. After incubation at 22-25 oC for 1 h, the plates
reference preparations. Such a model enables the analyst to are washed. A suitable solution of enzyme-conjugated anti-
determine whether the immunogenicity of the test mouse IgG antibody is added to each well anq incubated at
preparation is comparable to the reference vaccine, but do es 22-25 oC for 1 h. After washing, a chromogenic substrateis
not give information on linearity or parallelism of th~ dose- added from which the bound enzyme conjugate liberates a
response curves. chromophore that can be quantified by measurement of
For serological assays, imitable indicators to monitor test absorbance (2.2.25).
consistency are: Calculations The antibody titres in the sera of mice
- the mean and standard deviation of relative antibody immunised with reference and test vaccines are ca1culated for
levels or scores of the serum samples obtained after each acellular pertussis antigen using the reference
administration of a fixed dose of the vaccine reference antiserum, and from the values obtained the relative potency
preparation; of the test vaccine in relation to the reference vaccine is
- the antibody levels or scores of run controls (reference ca1culated by the usual statistical methods (5.3).
antiserum and negative serum samples); The assay is not valid unless:
- the ratio of antibody levels or scores for the reference - the confidence limits (P = 0.95) are not less than
antiserum to the serum samples corresponding to the 50 per cent and not more than 200 per cent of the
reference vaccine. relative potency estimate for each acellular pertussis
Where a single-dilution assay is used, production and test antigen;
consistency over time are monitored via suitable indicators - the statistical analysis shows a significant slope and no
and by carrying out a full multiple-dilution assay periodically, deviation from linearity and parallelism of the dose-
for example every 2 years. response curves (chapter 5.3 describes possible alternatives
if significant deviations are observed).
2014 Appendix XIV K V -A431
METHOD B. SEROLOGY IN GUINEA-PIGS The assay is not valid unless:

Selection and distribution of the test animals U se in - the confidence limits (P = 0.95) are not less than
the test healthy guinea-pigs from the same stock, each 50 per cent and not more than 200 per cent of the
weighing 250-350 g. Use guinea-pigs of the same sex or with relative potency estimate for each acellular pertussis
males and females equally distributed between the graups. antigen;
Distribute the guinea-pigs in not fewer than 6 equal groups; - the statistical analysis shows a significant slope and no
use groups containing a number of animals sufficient to deviation from linearity and parallelism of the dose-
obtain results that fulfil the requirements for a valid assay response curves (chapter 5.3 describes possible alternatives
prescribed below. During validation studies a further group if significant deviations are observed).
of guinea-pigs is used as a negative control by injecting the
The following section is published for infonnation.
animals with diluent alone.
Reference vaccine A batch of vaccine shown to be Assay 01 pertussis vaccine (acellular): guidelines
effective in clinical trials or a batch representative thereof is METHOD B. DETERMINATION OF ANTIBODIES IN
used as a reference vaccine. For the preparation of a GUINEA-PIGS
representative batch, strict adherence 10 the praduction The ELISA shown below is given as an example of an
process used for the batch tested in clinical trials is immunochemical method that has been found to be suitable.
necessary. The stability of the reference vaccine shall be Determination of antibody titre by ELISA method for
monitored and documented. pertussis toxin (PT), filamentous haemagglutinin
Reference antiserum An in-house guinea-pig reference (FHA), fimbrial agglutinogens (Firo 2/3) and pertactin
antiserum of assigned activity is used in the test and serves (PRN) 2-fold dilutions of sera fram test and reference
as the basis for calculation of the antibody levels in the test vaccines are made on EUSA pIates coated with acellular
sera. pertussis antigens (PRN, PT, FHA or Fim 2/3) . A guinea-
Dilution ofthe test and reference preparations Using a pig reference antiserum and a negative guinea-pig serum are
9 gIL solution of sodium chloride R as diluent, 'prepare serial included on each plate. Peroxidase-conjugated rabbit or goat
dilutions of the vaccine to be examined and the reference antibody directed against guinea-pig IgG is added, followed
preparation; series differing by 2.5- 10 5-fold steps have been by a peroxidase substrate. Optical density is measured and
found to be suitabIe. Use not fewer than 3 dilutions within the relative antibody titre is ca1culated by the usual statistical
the range found 10 be suitable for all the components in the methods (5.3).
vaccine 10 be examined. Use the dilutions for immunisation Reagents and equipment:
preferably within I h of preparation. Allocate I dilution to - ELISA plates: 96 wells, columns 1-12, rows A-H.
each group of guinea-pigs.
- Reference antiserum (guinea-pig).
Immunisation Inject subcutaneously into each guinea-pig
1.0 mL of the dilution allocated to its group. - Peroxidase conjugate. Peroxidase-conjugated rabbit or goat
antibody directed against guinea-pig IgG.
Blood sampling 35-42 days (5-6 weeks) after
immunisation, take a blood sample from each vaccinated and - Eordetella pertussis antigens (PRN, PT, FHA or Fim 2/3).
negative control guinea-pig using a suitable method. Store - Carbonate coating buffer pH 9.6. Dissolve 1.59 g of
the sera at -20 oC until used for antibody determination. anhydrous sodium carbonate R and 2.93 g of sodium
Avoid frequent freezing and thawing of serum samples. hydrogen carbonate R in 1000 mL of water R. Distribute
Antibody determination Assay the individual sera for into 150 mL bottles and sterilise by autoclaving at 121 ce
content of specific antibodies to each acellular pertussis for 15 mino
antigen using a validated method such as the EUSA test - Phosphate-buffered saline pH 7.4 (PES). Dissolve with
shown below. stirring 80.0 g of sodium chloride R, 2.0 g of potassium
ELISA test Suitable 96-well microtitre plates are coated dihydrogen phosphate R, 14.3 g of disodium hydrogen
with the purified antigens (e.g. pertussis toxin (PT), pertactin phosphate dihydrate R and 2.0 g of potassium chloride R in
(PRN), filamentous haemagglutinin (FHA) and/or fimbrial . 1000 mL of water R. S10re at room,temperature to
agglutinogens (Fim 2/3)) representing components in the prevent crystallisation. Dilute 10-fold with water R before
combined vaccine at a concentration of 200-400 ng per well. use.
After washing, unreacted sites are blocked by incubating the - Citric acid solution. Dissolve 10.51 g of citric acid R in
plates with a suitable blocking buffer and then washed. 1000 mL of water R and adjust 10 pH 4.0 with a 400 gIL
2-fold dilutions of sera from individual guinea-pigs solution of sodium hydroxide R.
immunised with test or reference vaccines are made on the - Washing buffer. PBS containing 0.5 gIL of polysorbate 20 R.
plates. Reference antiserum is included on each plate. After - Dzluent block buffer. PBS containing 0.5 gIL of polysorbate
incubation at 37 oC for 1 h, the plates are washed. 20 R and 25 gIL of dried skimmed milk.
A suitable solution of enzyme-conjugated anti-guinea-pig IgG - Peroxidase substrate. Shortly before use, dissolve 10 mg of
antibody is added to each well and incubated at 37 oC for
diammonium 2,2'-azinobis (3 -ethylbenzothiazoline-6-
1 h. After washing, a chromogenic substrate is added from sulfonate) R (ABTS) in 20 mL of the citric acid solution.
which the bound enzyme conjugate liberates a chromophore Immediately before use add 5 ¡lL of strong hydrogen
that can be quantified by measurement of absorbance
peroxide solution R.
(2.2.25).
Method The description below is given as an example of a
Calculations The antibody titres in the sera of guinea-pigs suitable plate layout but others may be used. Wells lA-H are
immunised with reference and test vaccines are calculated for used for negative control serum. Wells 2-12 A-H are used
each acellular pertussis antigen using the reference
for guinea-pig reference antiserum (usually in 2 positions)
antiserum, and from the values obtained the relative potency and individual sera from guinea-pigs immunised with the test
of the test vaccine in relation to the reference vaccine is or reference vaccine.
ca1culated by the usual statistical methods (5.3),
Coat each well of the EUSA plates with 100 J.lL of the obtain valid results for all 3 serotypes . The number of
appropriate antigen solution (PT, FHA and Fim 2/3 at animals per group must be sufficient ro obtain results that
2 J.lg/mL and PRT at 4 J.lg/mL, in carbonate coating buffer meet the validity criteria; groups of 10 rats are usually
pH 9.6). AlIow ro stand ovemight at 4 oC in a hum id sufficient, although valid results may be obtained with fewer
atmosphere. To avoid temperature gradient effects, do not animals per group. If animals of different sex are used, males
stack more than 4 plates high. On the following day, wash and females are evenly distributed between all groups.
the plates thoroughly with the washing buffer. Block the A weight range of 175-250 g has been found to be suitable.
plates by addition of 150 J.lL of the diluent block buffer to An inoculum of 0.5 mL per rat is used. The dose range is
each well. Incubate in a humid atmosphere at 37 oC for 1 h. chosen such that a dose response ro all 3 poliovirus types is
Wash the plates thoroughly with the washing buffer. Place obtained. Bleed the animal s after 20-22 days. Neutralising
100 ~lL of the diluent block buffer in each well of the plates, titres against all 3 poliovirus types are measured separately
except those of row A. Prepare suitable dilutions of using 100 CCID so of the Sabin strains as challenge viruses,
individual test and reference vaccine serum samples, Vero or Hep2 as indicaror cells, and neutralisation conditions
reference antiserum and negative control serum samples. of 3 h at 35-37 oC followed by 18 h at 2-8 oC where
Allocate the negative control serum ro column 1, the necessary for consistency of results. Results are read following
reference antiserum to at least 2 other columns and fixation and staining after 7 days of incubation at 35 oC.
individual tes~ and reference vaccine sera to the remaining For a valid antibody assay, the titre of each challenge virus
columns and add 100 J.lL of each serum to the first 2 wells of must be shown ro be within the range 10 CCIDso to 1000
the column to which it is allocated. Using a multichannel CCIDso and the neutralising antibody titre of a control
micropipene, make 2-fold serial dilutions from row B down serum must be within 2 twofold dilutions of me geometric
the plate ro row H, by transferting 100 J.lL from one well to mean titre of the serum. The potency is calculated by
the next. Discard 100 J.!L from the last row so that all wells comparison of the proportion of responders for the vaccine to
contain 100 J.lL. Incubate at 37 oC for 2 h. Wash thoroughly be examined and the reference vaccine by the pro bit method
with the washing buffer. Prepare a suitable dilution of the or, after validation, using a parallel-line model. For the probit
peroxidase conjugate in the diluent block buffer and add method it is necessary to establish a cut-off neutralising
100 J.lL ro each well. Incubate at 37 oC in a humid antibody titre for each poliovirus type to define a responder.
atmosphere for 1 h. Wash the plates thoroughly with the Due to interlaboratory variation, it is not possible ro define
washing buffer. Add 100 J.lL of the peroxidase substrate to cut-offvalues that could be applied by alllaboratories.
each well. Allow to stand at room temperature, protected Rather, the cut-off values are determined for each laborarory
from light, for 30 mino Read the plates at 405 nm in the based on a minimum series of 3 tests with the reference
same order as the addition of substrate was made. vaccine. The mid-point on a IOg2 scale of the minimum and
maximum geometric mean titres of the series of 3 or more
7. In vivo assay of poliomyelitis vaccine
tests is used as the cut-off value. For each of the 3 poliovirus
(inactivated)
types, the potency of the vaccine is not significantly less than
(Ph. Eur. method 2.7.20) that of the reference preparation. The test is not valid unless:
The capacity of the vaccine to induce the formation of
- for both the vaccine ro be examined and the reference
neutralising antibodies is determined in vivo by one of the
vaccine, the EDsó lies between the smallest and the largest
following methods.
doses given to the animals;
Test in chicks or guinea pigs - the statistical analysis shows no significant deviation from
Prepare a suitable series of not fewer than 3 dilutions of the linearity or parallelism;
vaccine ro be examined using a suitable buffered saline - the confidence limits (P = 0.95) are not less than
solution. Distribute either guinea-pigs weighing 250-350 g or 25 per cent and not more than 400 per cent of the
3-week-old chicks into groups of 10, and allocate a group ro estimated potency.
each dilution of the vaccine. Inject intramuscularly into each
The following section is published for infoñnation.
animal 0.5 mL of the dilution intended for its group , Bleed
the animals after 5-6 days and separate the sera. Examine the Guideline on waiving ofthe in vivo assaY 'of
sera for the presence of neutralising antibodies, at a dilution poliomyelitis vaccine (inactivated) and its
of 1 in 4, to each of the human poliovirus types 1, 2 and 3. combinations
Mix 100 CCID so of virus with the dilution of serum and
incubate at 37 oC for 4.5-6 h. Keep at 5 ± 3 oC for 12-18 h This guideline applies to vaccines derived !rom wild strains of
where necessary for consistency of results . Inoculate the poliovirus. The validation described should be cam"ed out for each
product befare waiving of the in viv o assay, and should be
mixtures into cell cultures for the detection of unneutralised
virus and read the results up ro 7 days after inoculation. repeated wherever there is a subsLantial change to the
For each group of animals, note the number of sera that have manufactun'ng process that may affect the in vitro or in vivo
assays.
neutralising antibodies and calcula te the dilution of the
vaccine that gives an antibody response in 50 per cent of the The European convention on the protection of vertebrate
animals. Carry out in parallel a control test using a suitable animals used for experimental and other scientific purposes
reference preparation. The vaccine complies with the test if a requires that tests in animals shall not be carried out if a
dilution of 1 to 100 or more produces an antibody response scientifically satisfactory alternative is reasonably and
for each of the 3 types of virus in 50 per cent of the animals. practically available. The aim of this guideline is therefore to
promote waiving of the in vivo assay wherever it can be
Test in rats shown for a given product that the in vitra assay (D-antigen
A suitable in vivo assay method consists of intramuscular determination) gives sufficient assurance of satisfactory
injection into the hind limb(s) of not fewer than 3 dilutions potency for routine batch control.
of the vaccine to be examined and a reference vaccine, using
For the in vivo assay, the test in rats is considered to be the
for each dilution a group of 10 specific pathogen-free rats of
method of choice. For vaccines that are assayed using chicks
a suitable strain. Use of 4 dilutions is often necessary to
or guinea-pigs and that have an established record of Practical experience has shown that the results of the
production history, the in vivo assay may be waived if the rat calibration of antitoxins in Intemational Vnits (IV), for
assay is also applied to the batch es included in the validation example by comparison to intemational antitoxin standards,
study described below. For vaccines not yet approved, the depends on the irnmunochemical method used. For this
results of the rat assay on aH final bulks should be included reason, antitoxins used for the Ramon assay must be directly
in aH data generated for demonstration of consistency of calibrated against the internacional biological reference reagents
production before waiving of the in vivo assay. for diphtheria or tetanus toxoid ¡or fiocculation tests, using the
Once the in vivo assay has been waived, batches of vaccine principies described below. The concentration thus
will be relea sed on the basis of the in vitro assay, and the in determined may be indicated in Lf-equivalents per miHilitre
vivo assay should not be used as an altemative for the release (Lf-eq./mL) .
of a batch that fails the in vitro assay. Repetition of the in By definition, 1 Lf is the quantity of toxin or toxoid that
vitro assay may be performed according to an authorised flocculates in the shortest time with 1 Lf-eq. of specific
procedure. antitoxin.
PROCEDURE A range of volumes of the reference standard of antitoxin
The foHowing conditions should be met before performance adjusted to a concentration of 100 Lf-eq./mL is dispensed
of the validation stÚdy: into a series of, for example, 7 cm x 1 cm flocculation
- appropriate experience of the rat assay; tubes. A sufficient quantity of a 9 giL solution of sodium
chloride R is added to each tube to give a constant total
- full validation of the D-antigen assay (linearity,
volume of, for example, 1 mL. The test sample is diluted to
repeatability, intermedia te precision, accuracy and limits
give an expected concentration of approximately 50 LflmL,
of quantification);
and, for example, 1 mL aliquots of this dilution are
- establishment of acceptance criteria for the D-antigen dispensed into each of the tubes containing antitoxin.
assay based on a suitable number of consecutive final lots; The tubes are properly mixed by shaking, then placed in a
- establishment of production consistency on recent final water-bath at a constant temperature between 30 oC and
bulks using the currently approved in vivo assay; the final 52 oC, and observed at regular intervals for the first
bulks should correspond to the finallots used to establish appearance of floccules. This may require the use of a
the acceptance criteria for the D-antigen assay and should magnifying lens and strong illumination.
represent different inactivated harvests of each of the 3 The first and the second mixtures to flocculate are recorded
types of poliovirus. as well as the time taken for the first flocculation to appear.
The validation study should be performed on: 2 tubes may flocculate simultaneously.
- a final bulkllot that is representative of the current The first tube to flocculate is the one that contains the
production method; amount of antitoxin closest in equivalence to the amount of
- 2 sub-potent batches prepared, for example, by heating antigen in the sample. The antitoxin content of this tube can
normal vaccine or mixing it with heat-treated vaccine; be used to calculate the Lf value of the sample. If 2 tubes
the sub-potent batches should have expected titres of flocculate at the same time, the mean from the tubes are
about half that of the representative final bulkllot. given as the resulto
These batches are assayed using as reference standard a The time taken for the first tube to flocculate (Kf) is a useful
homologous production batch: indicator of the quality of the antigen. If at a given
temperature and concentration of toxoid and antitoxin the Kf
- by the currently approved in vivo assay for the vaccine;
value is increased compared with normal, this indicates that
- by the rat assay where this is not the currently approved in the antigen has been damaged. The Kf value may also
vivo assay; change with the quality of the antitoxin used.
- by the D-antigen assay.
Waiving of the in vivo assay is acceptable if the representative
Example
final ~ulkllot complies with the in vivo and in vÍlro assays and
the sub-potent batches fail to comply. If a sub-potent batch
fails to comply with the D-antigen assay but complies with Tub. A B e D E F
the in vivo assay, the latter may be repeated. Antitoxin added 40 45 50 55 60 65
(Lf-eq.)
8. Flocculation value (Lt) of diphtheria and tetanus Antitoxin added 0.40 0.45 0.50 0.55 0.60 0.65
toxins and toxoids (Ramon assay) (rnL)
Saline added 0.60 0.55 0.50 0.45 0.40 0.35
(Ph. Eur. method 2.7.27) (rnL)
The content of toxin or toxoid in a sample can be expressed Diluted sarnple l.0 l.0 1.0 1.0 l.0 1.0
as a flocculation value (Lf) using the Ramon assay. In this added
assay, antitoxin is added in increasing concentrations to a
series of tubes containing a constant amount of toxin or
If in this example the first tube to flocculate is tube C then
toxoid. At the equivalence point of toxinltoxoid and
the Lf value of the diluted sample is 50 LflmL. However, if
antitoxin, flocculation occurs in 1 or more tubes. The first
the first tube to flocculate is tube A or tube F this do es not
tube in which flocculation occurs is used to determine the Lf
indicate equivalence at that leve!. It would be necessary to
value of the sample.
perform a repeat test using either a different dilution of test
The Lf value of a toxin or toxoid is determined by the sample or selecting a different range of doses of reference
number of units of antitoxin that, when mixed with the antitoxin.
sample, produces an optimally flocculating mixture (Ramon
More precision can be obtained by making allowance for the
assay).
sequence of flocculation after the first tube. Thus, in the
example quoted, if the second tube to flocculate had been
V-A434 Appendix XIV L 2014
tube D, the final value for the diluted sample would be 52, The negative control group of mice is injected
whereas if the second tube to flocculate was tube B, the final intraperitoneally with an equivalent volume of diluent.
value would be 48. The test may be performed in duplica te If a reference group of mice is used, it may be injected with a
with slightly different dilutions of the test sample. reference pertussis toxin preparation (e.g. pertussis toxin BRP)
If there is no indication of the expected Lf value of the at adose previously set as the allowable upper limit of
sample available, it is advisable 10 obtain a rough estimate by pertussis toxin in the product according to historical safety
use of a wider range of antitoxin content in the tubes before data. Alternatively, a reference vaccine with established
proceeding to the final test. c1inical safety may be used instead of the reference toxin
Example preparation.
After 5 days, inject intraperitoneally into each mouse of each
Tube A B e D E F group the equivalent of 2 mg of histamine base in a volume
not exceeding 0.5 mL and observe for 24 h. The test is not
Antitoxin 20 30 45 70 100 150
content va lid if:
(Lf-eq.) - 1 or more mice in the negative control group die following
histamine challenge;
The level of toxin or toxoid and antitoxin concentration in - the histamine sensitivity do es not meet the defined lirnit
the test may be varied, but this will markedly affect the (e.g. at least 30 per cent of the mice die in the positive
flocculation time, so that at very low levels the test will take control group).
too long, whilst at a high concentration the onset of
If a reference group is not included in rhe assay, the vaccine
flocculation may be so rapid as to make it difficult to
complies with the test for residual pertussis toxin if no mice
distinguish the first and second tubes to flocculate .
die in the 1st group. If a vaccine lot fails the test, the test may
Assay of low concentrations by blend flocculation be repeated once with the same number of mice or with a
greater number and the results of the tests combined;
For very low concentrations, it is preferable to measure toxin
the vaccine complies with the test if the percentage of deaths
or toxoid by the method of blend flocculation. This involves
in the group given the vaccine does not exceed 5 per cent.
comparison of the Lf value of a known toxin or toxoid and
The vaccine complies with the test for irreversibility of
that of a mixture of the sample with that toxin or toxoid. pertussis toxoid if the 2nd group, given the vaccine incubated
When a toxin or toxoid with a known Lf value and a toxin or at 37 oC, also complies with these criteria.
toxoid with an unknown Lf value are flocculated together,
If a reference group is included, the vaccine complies with
the mixture will flocculate as the sum of their values if they
the test for residual pertussis toxin if the percentage of deaths
are homogeneous. If non-homogenous toxins or toxoids are
in rhe 1st group is not greater than that in the reference
mixed they will produce an aberrant pattern with 2
group . If a vaccine lot fails the test, the test may be repeated
flocculation maxima .
once with the same number of mice or with a greater
9. Residual Pertussis Toxin and Irreversibility o/ number and the results of the tests combined; the vaccine
Pertussis Toxoid complies with the test if the percentage of deaths in the
groups given the vaccine does not exceed the percentage of
(Ph. Eur. method 2.6.33) deaths in the reference groups. The vaccine complies with
This test is not necessaJy for the product obtained by genetic the test for irreversibility of pertussis toxoid if the 2nd group,
modijication. given the vaccine incubated at 37 cC, also complies with
Pertussis toxin BRP or an in-house toxin preparation these criteria.
calibrated in International Units against the BRP are suitable Alternatively, after validation;a histamine-sensitisation test
for use as a reference pertussis toxin preparation. based on body temperature measurements as end-points may
Mouse strain A suitable mouse strain has a toxin LDso be used instead of the lethal end-point test in mice.
between 6 IU and 50 IU.
U se equal groups of not fewer than 10 histamine-sensitive
mice of suitable strain, age and weight. For the test for
residual pertussis toxin, inject intraperitoneally into the 1st L. Nucleic Acid Amplification Techniques
group between 1 and 2 human doses of the vaccine stored at (Ph . Eur. method 2.6.21)
2-8 oc. For the test for irreversibility of pertussis toxoid,
inject intraperitoneally into the 2nd group between 1 and 2 1. Introduction
human' doses of the vaccine incubated at 37 oC for 4 weeks.
Nucleic acid amplification techniques are based on 2
The histamine sensitivity criteria must be set during a different approaches:
validation study using multiple doses of a reference toxin;
l . amplification of a target nucleic acid sequence using, for
a significant dose-response must be demonstrated. A suitable
dose of the reference toxin, chosen in the linear region of the example, polymerase chain reaction (PCR), ligase chain
dose-response curve and giving a positive response reaction (LCR), or isothermal ribonuc1eic acid (RNA)
amplification;
considered appropriate by the competent authority, is
subsequentIy included in each assay as the positive control to 2. amplificarion of a hybridisation signal using, for example,
demonstrate assay sensitivity. for deoxyribonucleic acid (DNA), the branched DNA
The positive control group of mice is injected with an (bDNA) method; in this case signal amplification is
achieved without subjecting the nucleic acid to repetitive
equivalent volume of reference pertussis toxin preparation
(e.g. pertussis toxin BRP) at adose that has been defined in cyc1es of amplification.
the validation stage as demonstrating the assay sensitivity, for In this general chapter, the PCR method is described as the
example adose causing death in at least 30 per cent of the reference technique. Alternative methods may be used, if
mlce. they comply with the quality requirements described below.
2014 Appendix XIV L V-A435
2. Scope - PCR amplification (amplified material is handled in a

This section establishes the requirements for sample elosed system);
preparation, in vitro amplification of DNA sequences and - post-PCR detection (the only area where the amplified
detection of the specific PCR product. With the aid of PCR, material is handled in an open system) .
defined DNA sequences can be detected. RNA sequences 5.2. SAMPLE PREPARATION
can also be detected following reverse transcription of the When preparing samples, the target sequence to be amplified
RNA to complementary DNA (cDNA) and subsequent needs to be efficiently extracted or liberated from the test
amplification. material in a reproducible manner and in such a way that
amplification under the selected reaction conditions is
3. PrincipIe of the method possible. A variety of physico-chemical extraction procedures
PCR is a procedure that allows specific in vitro amplification and/or enrichment procedures may be employed.
of segments of DNA or of RNA after reverse transcription Additives present in test material may interfere with PCR.
into cDNA. The procedures described under 7.3.2. must be used as a
Following denaturation of double-stranded DNA into single- control for the presence of inhibitors originating from the test
stranded DNA, 2 synthetic oligonueleotide primers of material.
opposite p~larity anneal to their respective complementary In the case of RNA-templates, care must be taken to avoid
sequences in the DNA to be amplified. The short double- ribonuclease activity.
stranded regions that form as a result of specific base pairing
5.3. AMPLIFICATION
between the primers and the complementary DNA sequence
border the DNA segment to be amplified, and serve as PCR amplification of the target sequence is conducted under
starting positions for in vitro DNA synthesis by means of a defined cyeling conditions (temperature profile for
heat-stable DNA polymerase. denaturation of double-stranded DNA, annealing and
extension of primers; incubation times at selected
Amplification of the DNA occurs in cyeles consisting of:
temperatures; ramp rates) . These depend on various
- heat denaturation of the nueleic acid (target sequence) parameters such as:
into 2 single strands;
- the length and base composition of primer and target
- specific annealing of the primers to the target sequence sequences;
under suitable reaction conditions;
- the type of DNA polymerase, buffer composition and
- extension of the primers, which are bound to both single reaction volume used for the amplification;
strands, by DNA polymerase at a suitable temperature
- the type of thermocyeler used and the thermal .
(DNA synthesis).
conductivity rate between the apparatus, reaction tube and
Repeated cycles of heat denaturation, primer annealing and reaction fluid.
DNA synthesis results in an exponential amplification of the
5.4. DETECTION
DNA segment limited by the primers .
The amplicon generated by PCR may be identified by size,
The specific PCR product known as an amplicon can be
sequence, chemical modification or a combination of these
detected by a variety of methods of appropriate specificity
parameters. Detection and characterisation by size may be
and sensitivity.
achieved by gel electrophoresis (using agarose or
Multiplex PCR assays use several primer pairs designed for polyacrylamide slab gels or capillary electrophoresis) or
simultaneous amplification of different targets in one column chromatography (for example, liquid
reaction. chromatography). Detection and characterisation by sequence
composition may be achieved by the specific hybridisation of
4. Test material probes having a sequence complementary to the target
Because of the high sensitivity of PCR, the samples must be sequen ce or by eleavage of the amplified material reflecting
protected against external contamination with target target-specific restriction-enzyme sites. Detection and
sequences. Sampling, storage and transport of the test characterisation by chemical modification may be achieved
material are performed under conditions that minimise by, for example, incorporation of a fluorophore into the
degradation of the target sequence. In the case of RNA target amplicons and subsequent detection of fluorescence following
sequences, special precautions are necessary since RNA is excitation.
highly sensitiv'e to degradation by ribonueleases. Care must Detection of amplicons may also be achieved by using probes
be taken since sorne added reagents, such as anticoagulants labelled to permit a subsequent radioisotopic or irnmuno-
or preservatives, may interfere with the test procedure. enzyme-coupled detection.
5. Test method 6. Evaluation and interpretation of results
5.1. PREyENTION OF CONTAMINATION
A valid result is obtained within a test only if the positive
The risk of contamination requires a strict segregation of the control(s) is unambiguously positive and the negative
areas depending on the material handled and the technology control(s) is unambiguously negative. Due to the very high
used. Points to consider inelude movement of personnel, sensitivity of the PCR method and the inherent risk of
gowning, material flow and air supply and decontamination contamination, it is necessary to confirm positive results by
procedures. repeating the complete test procedure in duplicate, where
The system should be sub-divided into compartments such possible on a new aliquot of the sample. The sample is
as: considered positive if at least one of the repeat tests gives a
- master-mix area (area where exelusively template-free positive resulto As soon as a measurable target threshold is
material is handled, e.g. primers, buffers, etc.); defined, a quantitative test system is required.
- pre-PCR (are a where reagents, samples and controls are
handled);
7. Quality assurance 7.3.3. Threshold contml

7.1. VALIDATION OF THE PCR ASSAY SYSTEM The threshold control for quantitative assays is a test sample
The validation programme must include validation of with the analyte at a concentration that is defined as the
instrumentation and the PCR method employed. Reference threshold not to be exceeded. It contains the analyte suitably
should be made to the IeH guidelines (topic Q2B) Validation calibrated in International Units and is analysed in parallel in
of Analytical Method: Methodology. each run of a quantitative assay.
Appropriate official working reference preparations or 7.4. External quality assessment
in-house reference preparations calibrated against Participation in external quality assessment programmes is an
International Standards for the target sequences for which important PCR quality assurance procedure for each
the test system wilI be used are indispensable for validation of laborarory and each operaror.
a PCR test. The following sections are published for information.
7.1.1 . Determination of the positive cut-off point VALIDATION OF NUCLEIC ACID
During validation of qualitative tests, the positive cut-off AMPLIFICATION TECHNIQUES (NAT) FOR
point must be determined. The positive cut-off point is THE DETECTION OF HEPATITIS C VIRUS
defined as the minimum number of target seqúences per (HCV) RNA IN PLASMA POOLS: GUIDELINES
volume sample that can be detected in 95 per cent of test 1. Scope
runs. The positive cut-off point depends on interrelated
The majority of nucleic acid amplification analytical
factors such as the volume of the sample extracted and the
procedures are qualitative (quantal) tests for the presence of
efficacy of the extraction methodology, the transcription of
nucleic acid with sorne quantitative tests (either in-house or
the target RNA into cDNA, the amplification process and
commercial) being available . For the detection of HCV RNA
the detection.
contamination of plasma pools, qualitative tests are adequate
To define the detection limit of the assay system, reference and may be considered ro be a limit test for the control of
must be made to the positive cut-off point for each target impurities as described in the Pharmeuropa Technical Guide
sequence and the test performance aboye and below the for the elaboration of monographs, December 1999, Chapter
positive cut-off point. III 'Validation of analytical procedures'. These guidelines
7.1.2. Quantitative assay systems describe methods to validate only qualitative nucleic acid
For a quantitative assay, the folIowing parameters are amplification analytical procedures for assessing HCV RNA
determined during validation: accuracy, precision, specificity, contamination of plasma pools. Therefore, the
quantitatiol;l limit, linearity, range and robusrness. 2 characteristics regarded as the most important for
7.2. QUALITY CONTROL OF REAGENTS validation of the analytical procedure are the specificity and
All reagents crucial for the methodology used have to be the detection limito In addition, the robustness of the
controlIed prior to use in routine applications. Their analytical procedure should be evaluated.
acceptance/withdrawal is based on pre-defined quality However, this document may also be used as a basis for the
criteria. validation of nucleic acid amplification in general.
Primers are a crucial component of the PCR assay and as For the purpo.se of this document, an analytical procedure is
such their design, their purity and the validation of their use defined ~s the complete procedure from extraction of nucleic
in a PCR assay require careful attention. Primers may be acid to detection of the amplified products.
modified (for example, by conjugation with a fiuorophore or Where commercial kits are used for part or alI of the
antigen) in order to permit a specific method of detection of analytical procedure, documented validation points already
the amplicon, provided such modifications do not inhibit covered by the kit manufacturer can substitute for the
accurate and efficient amplification of the target sequence. validation by the user. Nevertheless, the performance of the
7.3 . RUN CONTROLS kit with respect ro its intended use has to be demonstrated by
7.3.1 . External cóntrols the user (e.g. detection limit, robustness, cross-
In order to minimise the risk of contamination and to ensure contamination) .
adequate sensitivity, the following external control s are 2. Specificity
included in each PCR assay:
Specificity is the ability ro assess unequivocalIy nucleic acid
- positive control: this contains a defined number of target- in the presence of components that may be expected to be
sequence copies, the number being close to the positive presento
cut-off value, and determined individualIy for each assay
The specificity of nucleic acid amplification analytical
system and indicated as a multiple of the positive cut-off
pro ce dures is dependent on the choice of primers, the choice
value of the assay system;
of probe (for analysis of the final product) and the stringency
- negative control: a sample of a suitable matrix already of the test conditions (for both the amplification and the
, proven to be free of the target sequences. detection steps) .
7.3.2. Interna! control When designing primers and pro bes, the specificity of the
Internal controls are defined nucleic acid sequences primers and probes ro detect only HCV RNA should be
containing, unless otherwise prescribed, the primer binding investigated by comparing the chosen sequences with
sites. Internal controls must be amplified with defined sequences in published data banks. For HCV, primers (and
efficacy, and the amplicons must be clearly discernible. probes) wilI normally be chosen from areas of the 5' non-
Internal controls must be of the same type of nucleic acid coding region of the HCV geno me which are highly
(DNNRNA) as the material to be tested. The internal conserved for all genotypes.
control is preferably added to the test material before The amplified product should be unequivocalIy identified by
isolating the nucleic acid and therefore acts as an overall using one of a number of methods such as amplification with
control (extraction, reverse transcription, amplification, nested primers, restriction enzyme analysis, sequencing, or
detection) .
hybridisation with a specific probe.
2014 Appendix XIV L V-A437
In order to validate the specificity of the analytical procedure, The evaluation of robustness should be considered during
at least 100 HCV RNA-negative plasma pools should be the development phase. It should show the reliability of the
tested and shown to be non-reactive. Suitable samples of analytical procedure with respect to deliberate variations in
non-reactive pools are availabJe from the European method parameters. For NAT, sma11 variations in the
Directorate for the Quality of Medicines & HealthCare method parameters can be crucial. However, the robustness
(EDQM) . of the method can be demonstrated during its development
The ability of the analytical procedure to detect a11 HCV when sma11 variations in the concentrations of reagents
genotypes wi11 again depend on the choice of primers, probes (e.g. MgCI 2 , primers or dNTP) are tested. To demonstrate
and method parameters. This ability should be demonstrated robustness, at least 20 HCV RNA negative plasma pools
using characterised reference panels. However, in view of the (selected at random) spiked with HCV RNA to a final
difficulty in obtaining samples of sorne genotypes concentration of 3 times the previously determined
(e.g. genotype 6), the most prevalent genotypes 95 per cent cut-off value should be tested and found positive.
(e.g. genotypes 1 and 3 in Europe) should be detected at a Problems with robustness may also arise with methods that
suitable level. use an initial ultracentrifugation step prior to extraction of
the viral RNA. Therefore, to test the robustness of such
3. Detection limit methods, at least 20 plasma pools containing varying levels of
The detection limit of an individual analytical procedure is HCV RNA, but lacking HCV-specific antibodies, should be
the lowest amount of nucJeic acid in a sample that can be tested and found positive.
detected but not necessarily quantitated as an exact value. Cross-contamination prevention should be demonstrated by
The nucJeic acid amplification analytical procedure used for the accurate detection of a panel of at least 20 samples
the detection of HCV RNA in plasma pools usua11y yields consisting of alternate samples of negative plasma pools and
qualitative results. The number of possible results is limited negative plasma pools spiked with high concentrations of
to 2: either positive or negative. Although the determination HCV (at least 10 2 times the 95 per cent cut-offvalue or at
of the detection limit is recommended, for practical purposes, least 104 IU/mL).
a positive cut-off point should be determined for the nucJeic
acid amplification analytical procedure. The positive cut-off 5. Quality assurance
point (as defined in the general chapter 2.6.21) is the For biological tests such as NAT, specific problems may arise
minimum number of target sequences per volume sample that influence both the validation and the interpretation of
that can be detected in 95 per cent of test runs . This positive results. The test procedures must be described precisely in
cut-off point is influenced by the distribution of viral the form of standard operating procedures (SOPs). These
genomes in the individual samples being tested and by should cover:
factors such as enzyme efficiency, and can result in different - the mode of sampling (type ofcontainer, etc.);
95 per cent cut-off values for individual analytical test runs.
- the preparation of mini-pools (where appropriate);
In order to determine the positive cut-off point, a dilution
- the conditions of storage before analysis;
series of a working reagent or of the hepatitis e virus BRP,
which has been calibrated against the WHO HCV - the exact description of the test conditions, incJuding
International Standard 96/790, should be tested on different precautions taken to prevent cross-contamination or
days to examine variation between test runs. At least 3 destruction of the viral RNA, reagents and reference
independent dilution series should be tested with a sufficient preparations used;
number of replica tes at each dilution to give a total number - the exact description of the appararus used;
of 24 test results for each dilution, to enable a statistical - the detailed formulae for calculation of results, incJuding
analysis of the results. statistical evaluation.
For example, a laboratory could test 3 dilution series on The use of a suitable run control (for example, an
different days with 8 replicates for each dilution, ·4 dilution appropriate dilution of hepatitis e virus BRP or plasma spiked
series on different days with 6 replica tes for each dilution, or with an HCV sample calibrated against the WHO HCV
6 dilution series on different days with 4 replicates for each International Standard 96/790) can be considered a
dilution. In order to keep the number of dilutions at a satisfactory system-suitability check and ensures that the
manageable level, a preliminary test (using, for example, log reliability of the analytical procedure is maintained whenever
dilutions of the plasma pool sample) should be carried out in used.
order to obtain a preliminary value for the positive cut-off Technical qualification An appropriate insta11ation and
point (i.e. the highest dilution giving a positive signal). operation qualification programme should be implemented
The range of dilutions can then be chosen around the for each critical piece of the equipment used.
predetermined preliminary cut-off point (using, for example, For confirmation of analytical procedure performance after a
a dilution factor of 0.5 log or less and a negative plasma pool change of critical equipment (e.g. thermocycJers), the change
for the dilution matrix). The concentration of HCV RNA should be documented by conducting a paraJlel test on 8
that can be detected in 95 per cent of test runs can then be samples of a plasma pool that is spiked with HCV RNA to a
calculated using an appropriate statistical evaluation. final concentration of 3 times the previously determined
These results may also serve to demonstrate the intra-assay 95 per cent cut-off value . AH results should be positive.
variation and the day-to-day variation of the analytical Operator qualification An appropriate qualification
procedure. programme should be implemented for each operator
4. Robustness involved in the testing. To confirm successful training each
operator should test at least 8 replicate samples of a plasma
The robustness of an analytical procedure is a measure of its pool spiked with HCV RNA to a final concentration of
capacity to remain unaffected by sma11 but deliberate 3 times the previously determined 95 per cent cut-off value.
variations in method parameters and provides an indication This test (8 replicate samples) should be repeated twice on 2
of its reliability during normal usage.
separate days, i.e. a total of 24 tests performed on 3 different assay precision); it is assessed by using 1 assay and testing
days. All results should be positive. 3 replicates of appropriate dilutions of a B 19V DNA-
positive sample suitably calibrated in International Units
VALIDATION OF NUCLEIC ACID and covering the whole quantitative range of the assay;
AMPLIFICA TION TECHNIQUES (NAT) FOR the coefficient of variation for the individual samples is
THE QUANTIFICATION OF B19 VIRUS (B19V) ca1culated (intra-assay variability);
DNA IN PLASMA POOLS: GUIDELINES - intermedia te precision expresses the intra-laboratory
variations (inter-assay precision) ; it is established by
1. Scope
assaying replica tes (as routinely used for the assay) of
The European Pharrnacopoeia requires that plasma pools appropriate dilutions of a B 19V DNA-positive sample
used for manufacture of certain products are tested for the suitably calibrated in InternationaI Units covering the
presence of B 19 virus (B 19V) DNA with a threshold whole quantitative range of the assay under different
concentration that must not be exceeded. In order to comply circumstances (e.g. different days, different analysts,
with these requirements, quantitative NAT tests are different equipment, different reagents); the coefficient of
preferred. The characteristics regarded as the most important variation for the individual samples is ca1culated;
fOr validation of the quantitative NAT procedure are
- reproducibility expresses the precision between different
accuracy, precision, specificity, quantitation limit, linearity
laboratories (inter-laboratory precision); it is assessed by
and range. In addition, the robustness of the analytical
participation in quantitative colIaborative studies on B 19V
procedure should be evaluated.
DNA-NAT assays, e.g. under the Proficiency Testing
This guideline describes methods to validate NAT analytical Scheme (PTS), including the comparative analysis of the
procedures for assessing B19V DNA contamination of obtained quantitative results, where appropriate.
plasma pools based on the ICH guidelines. However, this
docurnent may also be used as a basis for the validation of 4. Specificity
quantitative NAT in general. Specificity expresses the ability to assess unequivocally
For the purpose of this docurnent, an analytical procedure is nucleic acid in the presence of components that may be
defined as the complete procedure from extraction of nucleic expected to be presento The specificity of NAT analytical
acid to detection of the amplified products. procedures is dependent on the choice of primers, the choice
Where cornmercial kits are used for part or all of the of probe (for analysis of the final product) and the stringency
analytical procedure, documented validation points already of the test conditions (for both the amplification and the
covered by the kit manufacturer can substitute for the detection steps) .
validation by the user. Nevertheless, the performance of the When designing primers and probes, the specificity of the
kit with respect to its intended use has to be demonstrated by primers and probes to detect onIy human B19V DNA should
the user (e.g. ptecision, accuracy, range, robustness). be invéstigated by comparing the chosen sequences with
sequences in published data banks. There should be no
2. Accuracy major homology found with sequences unrelated to B 19V.
Accuracy expresses the closeness of agreement between the The amplified product should be unequivocally identified by
value that is accepted as either a conventional true value or using one of a number of methods such as amplification with
an accepted reference value and the value found . nested primers, restriction enzyrne analysis, sequencing, or
The accuracy of an assay is dependent on the calibration of hybridisation with a specific probe.
the assay and on the variance of the different assay steps.
In order to examine the specificity of the analytical
Though it is recommended to establish the accuracy across
procedure, at least 20 B 19V DNA-negative plasma pools
the specified range of the analytical procedure, the most
should be tested and shown to be non-reactive.
important assessment of accuracy is in the range of the
threshold concentration. In the case of B 19V NAT assays for Parvovirus B19 genotypes The International Committee
investigation of plasma pools it is recommended to assess the on Taxonomy of Virus es (ICTV) has classified
accuracy of the calibrated assay by assaying at least 5 representatives of the 3 genotypes as strains of human
concentrations (dilution factor of 0.5 log) of B19 VÚ'us DNA parvovirus B 19 . Genotype 1 represents prototype B 19V,
far NAT testing BRP or another material, suitably calibrated genotype 2 represents viral sequences like A6, and genotype
in International Units against the actual WHO B19 DNA 3 represents V9-like sequences. By performing sequence
International Standard, covering the range of the currently alignment with respective B 19V genotype sequences available
recommended threshold concentration of 10.0 IUIJ.1L B 19V from nucleic acid sequence databases, primers and probes
DNA (e.g. 105 IU/rnL, 104 .5 IU/mL, 104 lU/mL, 10 3 . 5 should be designed to detect and quantify consistently the
IU/mL and 103 IU/mL), with at least 3 replicates for each different human parvovirus B 19 genotypes. Reference
dilution. Accuracy should be reported for the different material s should be used to check the approach chosen.
concentrations in terms of percentage determined compared Since biological reference preparations refiecting sorne
with the known amount of B19V DNA. Ir should refiect the genotypes might be difficult to obtain, respective plasmid
level of technology of the respective assays, which should also preparations or synthetic nucleic acids may aIso serve as a
be defined, for example in collaborative studies. characterised target sequence source. However, those cannot
be used to validate the extraction procedure.
3. Precision
5. Quantitation Limit
Precision expresses the closeness of agreement between a
series of measurements, obtained from multiple sampling of The quantitation limit is the lowest amount of nucleic acid in
the same homogenous sample. The precision is defined at 3 a sample that can be determined quantitatively with suitable
1evels: precision and accuracy. The quantitation limit of the B 19V
- repeatability express es the precision under the same NAT assay is assessed during the repeatability and
operating conditions over a short interval of time (intra- intermediate-precision studies by limiting dilution analysis .
2014 Appendix XIV M V-A439
The lowest concentration of target nucleic acids that is - the detailed formulae for calculation of results, including
quantitated with suitable precision and accuracy is defined. statistical evaluation.
The inclusion of an appropriate threshold control (for
6. Linearity example, plasma spiked with a B19V DNA sample suitably
The linearity of an assay is its ability ro obtain test results calibrated in International Units, such as B19 virus DNA for
that are directly proportional ro the concentration of the NAT testing BRP) is considered ro be a satisfactory system-
nucleic acid. The linearity of the B 19V NAT assay is suitability check and ensures that the reliability of the
assessed during the repeatability and intermediate-precision analytical procedure is maintained whenever used.
studies by testing replica tes of diluted samples with the Technical qualification An appropriate installation and
concentrations covering the whole quantitative range. operation qualification programme should be implemented
The interval between the upper and the lower concentration for each critical piece of the equipment used.
of the target nucleic acid where test results are directly For confirmation of analytical procedure performance after a
proportional to the concentrations is defined. change of critical equipment (e.g. thermocyclers), the change
should be documented by conducting a parallel test on 8
7. Range samples of a plasma pool that is spiked with a concentration
The range of an assay is the interval between the upper and of B19V DNA around the threshold concentration.
the lower concentration of nucleic 'acid in the sample for AII results should be acceptable and reflect the features of
which it has been demonstrated that the procedure has a the assay as determined during the validation phase.
suitable level of precision, accuracy and linearity. The range Operator qualification An appropriate qualification
of the B 19V NAT assay is assessed during the repeatability programme should be implemented for each operator
and intermediate-precision studies by testing replicates of involved in the testing. To confirm successful training, each
diluted samples. The interval between the upper and the operator should test, on 3 separate days, at least 8 replicate
lower concentration that can be expressed with an acceptable samples of a plasma pool that is spiked with a concentration
degree of accuracy and precision is defined. of B 19V DNA around the threshold concentration
(Le. a total of 24 samples). AII results should be acceptable
8. Robustness
and reflect the features of the assay as determined during the
The robusmess of an analytical procedure is a measure of its validation phase .
capacity to rema in unaffected by small but deliberate
variations in method parameters and provides an indication
of its reliability during normal usage. The evaluation of
robusmess should be considered during the development M. Assay of Interferons
phase. It should show the reliability of the analytical (Ph. Eur. method 5.6)
procedure with respect ro deliberate variations in method The following chapter is published for infonnation.
parameters. For NAT, small variations in the method
parameters can be crucial. Nom;theless, the robusmess of 1. Introduction
NAT can be demonstrated during the development ofthe Monographs on human interferons generally contain a
method when smaiI variations in the concentrations of bioassay based on the inhibitory activity of the interferon on
reagents, for example MgCIz, primers or dNTP, are tested. the cytopathic action of a virus on a cellline in culture.
To demonstrate robusmess, at least 20 B 19V DNA-negative In most cases, however, the virus, cell line and the assay
plasma pool samples spiked with B19V DNA at the details a~e not specified, in order ro allow the appropriate
threshold concentration should be tested and found ro have f1exibility, where the monograph covers more than one sub-
acceptable quantitative values. c1ass of interferon.
Cross-contamination prevention should be demonstrated by The present text is intended to provide outline information
the accurate detection of a panel of at least 20 ,samples for the analyst on how to design, optimise and valida te such
consisting of alterna te samples of plasma pools without B19V an assay once an appropriate combination of cellline and
DNA or with levels below the threshold concentration (lO cytopathic virus has been identified. A detailed procedure for
samples) and plasma pools spiked with high concentrations a particular cytopathic antiviral assay is described as an
of B 19V DNA (at least 102 times the threshold level, 10 example of a suitable method, together with information on
samples). other virus-cellline combinations and guidance on how to
adapt and validate the procedure for these other
9. Quality Assurance combinations.
For biological tests such as NAT, specific problems may arise
that may influence both the validation and the interpretation 2. Antiviral (cytopathic effect reduction) assays
of results. The test procedures must be described precisely in The antiviral assay of human interferons is based on the
the form of standard operating procedures (SOPs) . These induction of a cellular response in human cells, which
should cover: prevents or reduces the cytopathic effect of an infectious
- the mode of sampling (type of container, etc.); virus. The potency of interferon is estimated by comparing its
- the preparation of mini-pools by manufacturers (where protective effect against a viral cytopathic effect with the
appropriate); same effect of the appropriate reference preparation
calibrated in International Units.
- the conditions of srorage before analysis;
- the exact description of the test conditions including 3. Interferon assay using Hep2c cells and infectious
precautions taken to prevent cross-contamination or encephalomyocarditis virus
destruction of the viral nucleic acids, reagents and The antiviral assay of human interferons described is of the
reference preparations used; cytopathic effect reduction type. It uses human Hep2c cells
- the exact description of the apparatus used; infected by encephalomyocarditis virus (EMCV) to measure
V-A440 Appendix XIV M 2014
the potencies of different human interferon test preparations. pH 7.4 to 7.8 to obtain a maximum virus yield. Remove the
This assay has been used in three World Health Organisation culture fluid and store at approximately 4 oC.
(WHO) international collaborative studies of candidate Place the flasks at - 20 oC to freeze the cell monolayer.
International Standards for human interferon alpha, human Then thaw to room temperature. Add approximately 5 mL
interferon beta and human interferon gamma and has of culture medium and shake the flask to disrupt the cell
repeatedly been demonstrated to be sensitive, reliable and walls. Transfer the contents of each flask to the container of
reproducible for potency estimations of the different types of culture fluid. Transfer the culture fluid containing the
human interferon. EMCV to 50 mL plastic centrifuge tubes and centrifuge at
For the culture of mammalian cells, all procedures are approximately 500 g for about 10 min to remove cell debris.
carried out using standard operating procedures for the Dispense the cIarified culture fluid into glass screw-capped
maintenance of such celllines in culture. Volumes of bottIes, in quantities of 20 mL, 10 mL, 5 mL, 1 mL, 0.5 mL
reagents are indicated for cell cultures carried out in 75 cm 2 or 0.2 mL, as appropriate. Store at - 70 oC . Larger volumes
flasks . Other types of containers (flasks or plates) may be can be thawed, dispensed into smaller quantities and
used but volumes must be adapted accordingly. re-frozen when required. The EMCV stock will retain its
3.1. Maintenance and preparation 01 Hep2c cells original titre if stored permanently at approximately - 70 oC,
but repeated freeze-thaw cycIes or storage at higher
Hep2c cells are maintained and passaged in culture medium temperatures, e.g. at approximately - 20 oC, results in
A.
progressive loss of titre.
Cells are stored as frozen stocks using standard operating
procedures. Growing cells may be maintained in culture up 3.3. Assay procedure
to a permitted passage number of 30, after which new 3.3. 1. DETERMINATION OF THE DO SE-RESPONSE RANGE
cultures are established from frozen stocks. Preparation of the solutions
At the beginning of the assay procedure, harvest the cells Dilute the appropriate standard for interferon (for example a
from the flasks showing 90 per cent confluent monolayers specific WHO sub-type interferon standard) in culture
using the trypsin-treatment procedure described below. medium A, in 10-fold dose increments, to give doses
covering the range of 1000 - 0.001 IU/rnL. Carry out the
- Remove the culture medium from the flasks.
assay procedure in 96-well microtitre plates. To each well
- To each flask, add 5 mL of trypsin solution heated at add 100 j.iL of culture medium A. Add approximately
37 oC (a trypsin stock solution contains 4 mglmL of 100 j.iL of each dilution of the reference preparation to each
trypsin R and 4 mglrnL of sodium edetate R; irnmediately well except for those intended for virus controls. Using a
before use, dilute 50 times with phosphate buffered multichanne1 pipette set at 100 j.iL, mix the contents of the
saline). Swirl the capped flask to wash the cell monolayer. wells.
Remove the excess of trypsin solution.
Dispensing of the cell suspension
- Incubate the flasks for 5 min to 10 min at 37 oC. Pour the cell suspension of Hep2c cells, which has been
Microscopically or visually observe the cells for signs of adjusted to contain approximately 6 x 105 cells/mL of
detachment. When viewed microscopically, the cells culture medium A, into a plastic Petri-dish. Dispense the cell
appear rounded up or detached and free-floating. Shake suspension from the Petri-dish into each well of the
the ft.ask vigorously to detach all the cells, add microtitre plates, using a multichannel pipette set at 100 j.iL.
approximately 5 mL of culture medium A. Shake
vigorously to yield a suspension of single cells. Incubate the plates for about 24 h in an incubator set at
37 oC and 5 per cent CO 2 •
- To prepare the cell suspensions for the assay procedure,
carefully disperse the cells by pipetting up and down to Viral infection
disrupt cell aggregates, count the cells and resuspend at a At this stage, using an inverted microscope, check that the
concentration of 6 x 10 5 cells/mL. monolayers of Hep2c cells are confluent, that they show a
relatively even distribution of cells, that they have correct
3.2. Propagation 01 encephalomyocarditis virus morphology and that they are healthy.
Encephalomyocarditis virus is propagated in mouse L-929 Remove most of the culture medium from the wells by
cens in order to produce a stock of progeny virus . L-929 cells inverting the plate and shaking it and blotting on a paper
are maintained by trypsin treatment and passage as described towel (proceed in an identical way when discarding fluids
for Hep2c cells (NOTE: it may be necessary ro substitute from micro-titre plates as described later). Dilute the EMCV
neonatal calf serum with foetal bovine serum if the cells show poor stock with fresh culture medium A to a titre of approximately
growth). 3 x 10 7 PFU/mL (NOTE: each plate requires approximately
Take several flasks containing confluent cultures of L-929 20 mL of dz7uted virus, plus 5 per cent ro 10 per cent of extra
cells. Pour off the medium from the flasks. Inoculate with volume). Dispense the diluted suspension from a 9 cm sterile
2 mL of the EMCV suspension appropriately diluted in Petri-dish using a multichannel pipette set at 200 ¡.tL to all
culture medium B so that it contains approximately 2.5 x wells incIuding virus controls, but excIuding cel! controls.
108 plaque forming units (PFU) per millilitre. Each flask will Add approximately 200 j.iL of culture medium A without
contain 4-6 x 10 7 L-929 cells and therefore the multiplicity virus to each of the cell control wel!s.
of infection (m.oj.) will be approximately 10 PFU/ce11. Retum the plates to the incubator set at 37 oC and
Carefully swirl the virus suspension over the entire cell 5 per cent CO 2 for approximately 24 h.
monolayer and return the flasks to the incubator for
approximately 1 h. Maintain the medium at pH 7.4 to 7.8. Staining
Examine the plates microscopically to check that the EMCV
After adsorption of the EMCV, add approximately 40 mL of
has caused a cytopathic effect (c.p.e.) in the virus controls .
culture medium B to each flask and retum the flasks to the
The time interval for maximum c.p.e. may vary from one
incubator at 37 oC for about 30 h. Maintain the medium at
assay to the next because of inherent variation of Hep2c cel!s
to virus challenge over a given period of continuous of the cellline/virus combination is usually based on that
cultivation. which gives the most sensitive response to the interferon
Remove most of the culture medium from the wells by preparation to be assayed, and gives parallel responses when
discarding into an appropriate decontaminating solution comparing the test preparation and interferon standard.
(sodium hypochlorite is suitable). Dispense phosphate buffered 4.2. Choice 01 response
satine pH 7.4 R into each well. Discard the phosphate buffered The staining procedure described aboye measures remaining
saline pH 7.4 R into a decontaminating solution. Dispense viable cells. A number of other responses have been used,
into each well 150 flL of staining solution. Stain the cells for including methyl violet or crystal violet staining, or the
approximately 30 min at room temperature. Discard the thiazolyl blue (MTT) conversion procedure. In each case, the
staining solution into a decontaminating solution. Dispense method is selected on the basis of producing a suitably linear
approximately 150 flL of fixing solution. Fix for 10 min at and sensitive relationship between response colour and viable
room temperature. Discard the fixing solution into a cell count.
decontaminating solution and wash the cell monolayers by
immersing the assay plates in a plastic box containing 4.3. Statistical validation
running water. Discard the water and superficially dry the As with all parallelline bioassays, the assay must satisfY the
plates with paper rowels. Dry the assay pi ates at 20 cC to usual statistical criteria of linearity of response, parallelism
37 oC until all moisture has evaporated. and variance.
Add 150 flL of 0.1 M sodium hydroxide ro each well. Elute 4.4. Validation 01 assay layout
the stain by gentle agitation of the plates or by hitting them As with all microtitre plate assay procedures, attention must
against the palm of the hand. Make sure that the stain is be given ro validating the assay layout. In particular, bias due
evenly distributed in all wells before making to non-random pipetting order or plate edge effects must be
spectrophotometric readings. investigated and eliminated, by randomising the assay layout,
Read the absorbance at 610 nm to 620 nm, using a or by avoiding the use of edge wells.
microtitre plate reader, taking as a blank a well or a column
of wells containing no célls and approximately 150 ¡.tL of Reagents and culture media
0.1 M sodium hydroxide. Culture medium A (J Oper cent neonatal calj serum)
Estimate the concentrations of interferon standard that give RPMI 1640 culture medium, supplemented with 450 mL
the maximum and minimum reduction of cytopathic effect. antibiotics if necessary (penicillin 10 000 IU/mL;
This is the dose response corresponding to the working range streptomycin 10 ng/mL)
of the assay. L-Glutamine, 200 mM, sterile 5 mL
3.3.2. ASSAY PROCEDURE Neonatal calf serum 50 mL
Carry out the assay as described aboye, using: Culture medium B (2 per cent ¡oetal bovine serum)
- as test solutions, the substance to be examined, diluted in RPMI 1640 culture medium, supplemented with 490 mL
two-fold increments with culture medium A to give antibiotics if necessary (penicillin 10 000 IU/mL;
nominal concentrations covering the working range of the streptomycin 10 ng/mL)
assay, L-Glutamine, 200 mM, sterile 5 mL
- as reference solutions, the appropriate standard for Foetal bovine serum 10 mL
interferoll. (for example, a specific WHO sub-type
Staining solution
interferon) in culture medium A, diluted in two-fold
increments to give nominal concentrations covering the Naphthalene black 0.5 g
working range of the assay. Acetic acid, glacial 90mL
Sodium acetate, anhydrous 8.2 g
3.3.3. DATA ANALYSIS
Water ro 1000 mL
Results of the cytopathic effect reduction assay generally fit a
sigmoidal dos e-response curve, when the interferon Fixing solution
concentration (the log of the reciprocal of the interferon Formaldehyde, 40 per cent 100 mL
dilution) is plotted versus stain absorbance. Acetic; acid, glacial 90 mL
Plot the interferon concentration (log reciprocal of dilution) Sodium acetate, anhydrous 8.2 g
versus the stain absorbance for the interferon reference Water to 1000 mL
preparation and for the interferon test solutions. Using the
linear portion of the curve, calculate the concentration of
interferon in the sample by comparing the responses for test
and reference solutions, using the usual statistical methods
N1. Numeration of CD34/CD45+ Cells in
for a parallelline assay. Haematopoietic Products
4. Validation of other procedures (Ph. Eur. method 2. 7.23)
This chapter describes immunolabelling and analysis by fiow
4.1. Choice 01 cellline and virus cytomerry (2.7.24) ro determine the number of
A number of other combinations of cellline and virus have CD34/CD45 + cells contained in haematopoietic products.
been used in anti-viral assays for interferons. For example, The determination is carried out by a single platform method
EMCV has been used in combination with the A549 using calibrated fiuorospheres, after Iysis of the sample red
epithelial iung carcinoma cellline, Semliki Forest virus or blood cells if necessary.
Sindbis virus have been used with human fibroblasts, and
This method applies ro all types of preparations and whole
vesicular stomatitis virus has been used with either human
blood. However, its level of precision makes it particularly
diploid fibroblasts, the human amnion WISH cell line or the
suitable for preparations containing very low percentages of
Madin-Darby bovine kidney cellline. In each case the choice
CD34/CD45+ cells.
V-A442 Appendix XIV M 2014
Graft quality assessment by CD34/CD45+ cell A number of CD34/CD45+ cells counted;

enumeration B number of f1uorosphere singlets counted;
A variety of studies have established that the 1-3 per cent of e known f1uorosphere concentration.
cells in the bone marrow that express the CD34 cell surface
antigen are capable of reconstituting long-term, multilineage Gating strategies
haematopoiesis after myeloablative therapy. CD34/CD45+ The purpose of sequential gating is to select the population
cells are also found in the peripheral circulation of normal of interest and simultaneously minimise interference from
individuals but are extremely rare (0.01-0.1 per cent). debris and mature cells to which antibodies can bind non-
However, CD34/CD45+ cells may also be mobilised from specifically. If using a commercial kit, apply the gating
marrow to the peripheral circulation in greater numbers by recommended by the manufacturero If using an in-house
haemaropoietic cytokines such as granulocyte colony- assay, it is preferable ro apply a currently recommended
stimulating factor andlor chemotherapy. strategy. A gating strategy that uses light scattering
The technique used for enumeration of CD34/CD45+ cells parameters and CD34/CD45 f1uorescence will aid in the
accurate identification and enumeration of CD34/CD45+
must meet the following requirements:
cells.
- high sensitivity, since haematopoietic stem cells are rare
events; Number 01 events analysed
- accuracy, to provide clinically relevant results; A sufficient number of events are analysed to maintain
acceptable precision, for example not fewer than 100 CD34+
- reproducibility, to provide clinically reliable results;
events and not fewer than 60 000 CD45+ events; the total
- speed, ro provide real-time analysis. number of cells counted may be greater if the percentage of
Selection 01 parameters CD34 is 0.1 per cent or less.
The f10w cytometry assay uses commercially available, Specimen collection
directly conjugated f1uorochrome-labelled monoclonal Acid citrate dextrose (ACD) formula A is the anticoagulant
antibodies, routine staining and whole blood lysing used in apheresis procedures. This anticoagulant allows both
procedures, and a gating strategy using light scatter and an auromated leucocyte count and f10w cytometry evaluation
immunofluorescence analysis using a pan-CD45/CD34 ro be performed on the same specimen. Edetic acid (EDTA)
monoclonal antibody combination. is the anticoagulant of choice for peripheral blood sampling.
lt is possible to determine CD34/CD45+ cell viability by
Specimen transport
appropriate nucleic acid staining with a stain that do es not
eros s the intact cell membrane (for example, with Transporr conditions guarantee the physical and thermal
7-aminoactinomycin D). safety of samples.
Selection 01 monoclonal antibodies Specimen integrity and storage
CD34 antibodies Use class III CD34 antibodies that Fresh (less than 24 h old) apheresis products, whole blood
detect all glycosylation variants of the molecule (for example, samples, umbilical cord blood specimens or bone marrow
clone 8G12 or 581). To detect rare events, use an antibody samples can be processed. Old specimens (more than 24 h
conjugated ro the brightest f1uorochrome excitable using an old) and specimens that have been frozen and thawed are
argon laser-based f10w cytometer, for example phycoerythrin stained with a viability dye. On receipt, the temperature
(PE). within the package is verified.
CD45 antibodies Pan-CD45 antibodies that detect all
TECHNIQUE
isoforms and all glycoforms of this structure are required.
A CD45 antibody conjugated ro f1uorescein isothiocyanate Sample preparation
(PITC) f1uorochrome is generally used (for example, J33, Ensure that the concentration of leucocytes is suitable prior
HLel,2Dl). ro staining with monoclonal antibodies. If necessary, dilute
Isotypic or isoclonic controls A negative control is the sample with medium that is compatible with the product
analysed to detect any non-specific signal in the PE ro be tested and the Iysing system. Record the dilution factor.
f1uorescence region. If using an isotypic control (a lt is recommended ro perform the test with a negative
monoclonal antibody ro an irrelevant antigen of the same control.
isotype as the CD34 antibody employed), the PE-conjugated Flow cytometry analysis
isotype is combined with CD45-FITC (or PerCP). If using AUTOSTANDARDISATION
an isoclonic control, the unconjugated (in excess) and For analysis of cells labelled with a commercially available
PE-conjugated CD34 identical monoclonal antibody is kit, manufacturers have developed sorne quality tools for
combined with conjugated CD45. Altemative combinations setting the f10w cytometer. These settings are then
may be used. automatically transferred on protocol analysis of samples .
Absolute count 01 CD34/CD45+ Specific f1uorospheres are used to set the photomultiplier
Calibrated fluorospheres Depending on the technique tube (PMT) on target values, compensaríon is set and the
used, the intemal standard either consists of calibrated beads system is checked using a control preparation.
in suspension or is directly introduced into the associated SYSTEM SETTINGS
tubes by the manufacturer. - Discriminator/threshold: the forward angle light scatter
The absolute count of the CD34/CD45+ cells per microlitre threshold is set to exclude debris (low forward scatter) but
is calculated using the following expression: not small lymphocytes from the light-scatter plot.
- PMT high voltage settings: these must be consistent with
A cell-surface marker analysis and established within each
-xC laborarory so that negative and positive cell populations of
B
moderate antigen density can be distinguished;
PMT voltages are reviewed and adjusted periodically Greater specificity on the general class of HPCs and on their
according to standardised laboratory procedures. relative proliferative potential is provided by the time
- Compensation: this must be acceptable for the colour required to differentiate in vicro into mature cells. The time
spectra overlap (for example, FITC/PE) encountered in required by post-natal colony-forming cells to give rise to a
cell-surface marker analysis; colour compensation is colony formed of mature celIs in vitro is 10-14 days.
analysed and adjusted according to standardised
laboratory procedures. Quality assurance for a CFC assay
- Flow rate: this must be consistent with routine cell-surface It is paramount for the overall quality of the colony-forming
marker analysis. cell assay to apply a strictly standardised approach. It is
therefore recommended to carry out intra- and inter-
- Gating regions: the gating regions established for the
laboratory validations. The source of the materials, including
CD34/CD45 samples are maintained unaltered for the
reagents, growth factors and disposables, is identified.
analysis of the negative region.
The main factors affecting variability in the CFC assay are
Calculation oi absolute number oi CD34/CD45+ cells the number of cells plated and the identification of colonies.
The absolute number of CD34/CD45+ cells is calculated Up to 15 per cent intra-Iaboratory variability may be
using the following expression: observed for the same test. If it is necessary to evaluate the
number of colony-forming celIs in a purified cell population,
nxDxV it is possible to use a limiting dilution approach where the
number of wells positive for celI proliferation is measured
11 total number of CD34/CD45+ cells per microlitre; with an automated system.
D dilution factor;
V volume of the product to be tested, in microlitres . The other main source of variability stems from the use of
undefined materials (for example, foetal bovine serum or
Results are reported as both the percentage of CD34/CD45 + bovine serum albumin) in the CFC assay. These products
cells and the absolute number per microlitre. They may also derive from pools of source material s and provide a non-
be reported as the absolute number per kilogram of recipient specific stimulation of celIular proliferation. However, it is
body mass, where this is possible. not uncommon to have batches with particular characteristics
that selectively stimulate the proliferation of specific
haematopoietic Iineages.
FinalIy, a low level of endotoxins (Iess than 0.01 IU/mL or
N2. Colony-forming Cell Assay for les s than 0.01 IU/mg) in all the materials used for the
Human Haematopoietic Progenitor Cells clonogenic assay is advisable, as higher levels result first in a
progressive skewing of the haematopoietic lineages expression
in the cultures, and afterwards in a more general inhibition of
The haematopoietic system represents a continuum of cells
celI proliferation and clonogenesis.
whose phenotype and properties change as they progress
from stem cells to differentiated cells. CFC c1onogenic assay
Haematopoietic progenitor cells (HPCs) are capable of The CFC assay is based on the capacity of progenitor celIs to
forming colonies or 'cell clusters' in cultures grown in semi- form a colony when plated in a semi-solid medium or in a
solid media and are said to be 'clonogenic'. gel in the presence of specific growth factors. Different types
The determination of the number of colony-forming cells of semi-solid media may be used (for example,
(CFCs) in a cellular product is an indicator of the functional methylcellulose, colIagen, agar and plasma-clot) depending
capacity of the progenitor 'cells and is a predictor of
on the desired readout. CommercialIy available media usualIy
haematopoietic reconstitution. The measured number of give more reproducible results.
CFCs correlates with the minimum number of progenitors
present in the sample. MATERIAL S
A validation is perforrned at least for the following critical
Cell-surface markers materials.
The capacity of co;lony-forming cells to give rise to Growth factors Both multilineage (such as Kit-ligand or
haematopoietic colonies in vicro andlor to reconstitute the stem cell factor (SCF), interleukin-3, granulocyte-
haematopoietic system has been correlated with the macrophage colony-stimulating factor (GM-CSF)) and
expression of specific cell-surface antigens. The expression of lineage-specific (erythropoietin, granulocyte colony-
the membrane antigen CD34 is an accepted marker for most stimulating factor (G-CSF)) growth factors are required to
of the haematopoietic progenitors and stem cells. obtain the highest number of colonies from a cell suspension
containing a mixed population of HPCs.
Colony assay specificity Other media components Media may be supplemented
Colony-forming cells are identified with a nomenclature by serum (notably by foetal bovine serum) and/or albumin.
based on the Iineages of mature cells present in the colony CELL CULTURE
(for example, CFU-Mix, CFU-GEMM, CFU-GM, CFU-G, Cells The sample placed in culture must be representative
CFU-M, BFU-E, CFU-E, CFU-Meg) and are a population of the celIular product injected. CelI suspensions are required
of progenitors able to give rise to colonies containing one or for this assay. In the case of bone marrow aspirates, such
more Iineages of haematopoietic cells. No or low capacity for suspensions can be obtained by forcing the bone marrow
self-renewal has been ascribed to this population of human through a sieve or through progressively smaller calibre
HPCs compared with the most immature stem cells. needles. Repeated passages through a 21-gauge needle are
The amount and type of growth factors supplied during the usually sufficient to disperse cell clusters into a cell
culture modulate the type and size of colonies that will be suspension.
formed.
V -A444 Appendix XIV M 2014
PLATING AND SCORING thick slide and a coverslip mounted to delimit a chamber
The celIs diluted in the culture medium are mixed in the with a specific volume for each design oThe thick slide of
semi-solid medium. It is common to plate 1 mL of the the various haemocytometers consists of counting
mixture in an untreated sterile Petri dish (0 35 mm). chambers separated by deep gro oves to avoid cross-filling.
Because of the viscosity of the medium, the solution cannot The counting chamber is etched in the glass and contains
be plated with air displacement pipettes and the use of a grid which is specific for each model;
syringes equipped with large bore (::; 18-gauge) needles is - a light microscope - low power 10 x to 40 x
required. magnification;
The number of celIs to be plated depends on the HPC - pipettes of a suitable volume range.
concentrarion in the sample to be tested. So that no colony is The haemocytometer is used to quantify the number of cells
derived from 2 different HPCs, the number of celIs plated in a given solution by calculation of the cell concentration
must alIow between 40 and 80 colonies per plate (0 35 mm) per milIilitre (C) using the following expression:
to be counted. The 'target' number of colonies per plate may
be obtained either from the percentage of CD34+ (or a x IOn X d
concentrarion of CD34+ celIs/mL) determined by flow
cytometry (2.7.24) or from different dilutions of the celI
suspension (usually 2 concentrarions are tested). a number of cells counted;
d dilution factor (where applicable);
The plates are incubated in aerobic conditións with a carbon
n factor varying with the volume of the haemocytometer
dioxide concentration of 5 per cent, at 37 oC in a humid
chamber.
(saturated) atmosphere for 10-14 days, and the number of
colonies is then scored under an inverted microscope. Care It is possib1e to distinguish between mixed cell populations
must be taken when manipulating the dishes containing the provided they differ in size or pigmentation (for example,
colonies as the methylcelIulose-based medium is viscous but leukocytes and erythrocytes).
not jelIified. An inclined plate will result in mixed and Preparation of the counting chamber and analysis
'comet'-shaped colonies making the scoring likely to be Mount the coverslip (slightly moistened on the edges) on the
incorrecto slide. Move the coverslip back and forth over the slide,
IDENTIFICATION OF THE COLONIES pressing slightly on the sides. Prepare a suitable dilution of
The size and structure of the colonies depend on the type of the cell suspension in isotonic buffer or in haemolysis buffer.
mature celIs that are their constituents. 50 celIs per colony is Add an appropriate volume of the dilution to the counting
usualIy considered a minimum. The presence of chamber. The liquid is added ro the border of the coverslip
haemoglobinised celIs identifies progenitors of the erythroid and is drained inside the chamber by capillarity. Carefully
lineage. As the amount of mature celIs for each lineage place the haemocytomerer under the microscope and focus .
largely depends on the growth factors added to the cultures, Count the cells in a zone of rhe grid. Calculate the cell
performing differentiated counts is not recommended unless concentration in the diluted and original samples.
otherwise prescribed. To increase the accuracy of the measurement, it is important
EXPRESSION OF THE RESULTS to respecr the following basic precautions:
The results of CFC culture are usually expressed as the - use only suitably thickened coverslips;
arithmeric mean of the number of colonies counted in at - wherever possible, count more than 100 cells (if necessary,
least 3 plates in the test. The mean number of colonies is count more areas);
then related to 104 or 105 viable nucleated celIs placed in - where cell clustering is derected (i.e. the cell suspension is
culture. not monocellular), resuspend the cells before sampling
and count again;
- avoid underfilling or overflowing the chamber, otherwise
N3. Nucleated Cell Count and Viability the volume will no longer be accurate.
AUTOMATED COUNTING METHODS
The determination of the quality of celI suspensions requires Particle counters based on conductivity variation
accurate measurements of both celI concentration and Electronic particle counting devices measure the size and
percentage of vi~ble celIs. These data are essential to the number of particles in a solution.
decision-making process for preparing celIular products and Particle counters are calibrated before use with a solution of
for maintaining optimum culture conditions. The celI count particles of known concentration and size. To allow the
may be expressed as the number of celIs per volume of celI counting of larger particles, tubes fitted with differently
suspension and the celI viability as the number of viable cells calibrated orifices are available . These apparatuses do not
per volume of cell suspension. The celI-count procedure may allow the discrimination between dead and live cells. As cell
be performed manually (haemocytometer) or with an debris may also generate pulses that may cause errors,
automated apparatus (for example, particle counter, flow counters are also fitted with a threshold control allowing only
cytometer). Other methods than that described below may be larger particles to be counted.
used. The apparatus must be qualified for the counting of cellular
products (in terms oflinearity, accuracy, etc.).
Cell number
Particle counters based on flow cytometry (2.7.24)
MANUAL COUNTING The flow cytometer is calibrated with reference partic1es of
Description of the apparatus and test principie The known concentration and size to give an absolute cell
following materials are required: number per volume. However, a calibrating solution is no
- a haemocytometer: a specialised microscope counting longer necessary in instruments using 2 electrodes inserted in
chamber available in different designs . It consists of a the sampling chamber where the fixed size of the sampling
chamber and distance between the 2 electrodes allow the is generally performed with 7-aminoactinomycin D (7-AAD)
measurement of the content of a fixed volume. This type of or propidium iodide (PI) but other suitable dyes may also be
instrument rarely needs to be calibrated after the initial used.
setting. Dye 7-AAD and PI are given as examples of membrane-
impermeants that may be used as viability dyes.
Viability
7-AAD is an analogue of actinomycin D that contains a
This section applies to cen staining by viability dyes and substituted amino group at position 7 of the chromophore.
manual or automated analysis, under a light microscope or It intercalates between cytosine and guanine DNA bases.
by ftow cytometry, of a cen suspension in order to determine The spectral properties of 7-AAD make this molecule
the percentage of viable censo particularly suitable for ftow-cytometry analysis.
Depending on the type of cens and the method used, the The maximum absorption ofthe 7-AADIDNA complex is
results may differ. situated in the green spectral region and is thus suitable for
MANUAL DYE-EXCLUSION METHOD an argon laser-equipped cytometer (excitation wavelength of
Test principIe This test is based on the exclusion ofthe 488 nm) . The deep red ftuorescence emission of the 7-AAD
dye from viable cens whereas dead or damaged cens absorb viability dye (635 nm to 675 nm) eases the use of the probe
the dye and are coloured. It provides information on the in combination with ftuorescein isothiocyanate (FITC) and
cytoplasmic membrane integrity but its results do not phycoerythrin (PE)-conjugated antibodies, because in
necessarily reftect cen functionality. Recently trypsinised or contrast to PI, the 7-AADIDNA complex shows minimal
thawed viable cells may have leaky membranes, causing them overlap with FITC and PE.
to absorb the dye. PI binds to double-stranded DNA by intercalating between
Dye Trypan blue is the stain most commonly used to bases with little or no sequence preference and with a
distinguish between viable and non-viable cens, but other stoichiometry of 1 dye molecule per 4-5 DNA base pairs.
suitable dyes such as erythrosin B or nigrosin may also be Once the dye is bound to nucleic acids, its ftuorescence is
used. It is an acid dye (Mr 961), an anion with 4 sulfonate enhanced 20- to 30-fold, the ftuorescence excitation
groups that can easily bind to proteins; therefore the protein maximum is shifted around 30-40 nm towards the red and
concentration of the preparation to be tested must be as low the ftuorescence emission maximum (615 nm) is shifted
as possible. around 15 nm towards the blue . Although its absorptivity is
quite low, PI exhibits a sufficiently large Stokes shift to allow
Test conditions Dye fixation is strongly inftuenced by pH,
simultaneous detection of nucleic acids and ftuorescein-
within a range of 6.6 to 7.6. Fixation is optimal at pH 7.5 .
labened antibodies, provided that the suitable optical filters
The other conditions, such as the dye concentration and the
are used .
staining time are validated.
Storage conditions of nucleic acid dye solution
Storage conditions ofthe dye Generanya 0.4 or
5 ± 3 oC.
0.5 per cent trypan blue solution in sterile phosphate-
buffered saline is used. Store protected from light and airo Test preparation and analysis In the case of
haematopoietic cens, the dye may be added after CD45
Test preparation and analysis Stain the cen suspension
labening to obtain a better separation of cens from debris
at the required dilution (usually in phosphate-buffered saline)
and platelets with a side scatter (SS)/CD45+ gating region.
with, for example, a trypan blue solution having a final
The incubation conditions of the cen suspension with the
concentration of 0.1 to 0.2 per cent. Mix gently. Incubate
dye are validated previously.
for not more than 2-4 min at room temperature. Mix gently
and place a suitable volume in a counting chamber. Count Incubation is performed at room temperature protected from
without delay. light. Where necessary, lysis of red blood cens is performed
using, for example, ammonium chloride. If not, add buffer
Determine the percentage of viable cens from the ratio of the
alone .
number of unstained cens to the total number of cens under
a light microscope, considering all stained cens as dead cells . Percentages of viable cells are directly given by the ftow
Viability (JI) is calculated as a percentage using the fonowing cytometer and deduced from the analysis of positive cells
expression: (dead cens) in the SS/7-AAD or SS/PI cytogram (dot plots).
Positive controls may consist of stabilised cells (dead cens)
n mixed with fresh viable cells at a target value.
N x 100
Digital imaging of stained cells Digital imaging allows
the automation of dye-exclusion methods . The cell
n = number of unstained (viable) cens;
suspension and viability-dye solution are directly mixed by a
N = total number of cells (stained and unstained).
machine. The system, which allows sample aspiration,
It is essential that the incubation time be not more than reagent handling, and subsequent instrument cleaning is funy
4 min as the number of stained cens may increase automated. Once the cenular suspension has been aspirated
significantly afterwards. For a new determination, it may and mixed with the dye solution, it is pumped to the ftow
therefore be necessary to prepare a new test. cell for imaging. The stained cen suspension is aspirated
AUTOMATED METHODS through a chamber where stroboscopic light allows a camera
to photograph the ftowing cells. The images are digitalised
Flow cytometry and the number of dead or live cens counted by the
Test principIe The test is based on the ability of certain software.
dyes to cross damaged membranes and bind to DNA by
intercalating between bases so that dead cens may ftuoresce
and be detected by ftow cytometry (2. 7.24) . Non-viable cens
are evaluated and discriminated by focusing on positive
staining whereas viable cens remain unstained. This analysis
V -A446 Appendix XV 2014
Control cells A quantity of cells set aside, at the time of

Appendix XV virus inoculation, as uninfected ceH cultures. The uninfected
cells are incubated under similar conditions to those used for
the production cell cultures.
Production and Testing of Vaccines Single harvest Material derived on one or more occasions
A. Terminology used in Monographs on from a single production cell culture inoculated with the
same working seed lot or a suspension derived from the
Biological Products working seed lot, incubated, and harvested in a single
(Ph. Eur. methad 5.2.1) production runo
For sorne items, altemative terms commonly used in Monovalent pooled harvest Pooled material containing a
connection with veterinary vaccines are shown in parenthesis. single strain or type of micro-organism or antigen and
Seed-Iot system A seed-Iot system is a system according derived from a number of eggs, cell culture containers etc.
to which successive batches of a product are derived from that are processed at the same time.
the same master seed lot. For routine production, a working Final bulk vaccine Material that has undergone all the
seed lot may be prepared from the master seed lot. steps of production except for the final filling. It consists of
The origin and the passage history of the master seed lot and one or more monovalent pooled harvests, from cultures of
the working seed lot are recorded. one or more species or types of micro-organism, after
Master seed lot A culture of a micro-organism distributed c1arification, dilution or addition of any adjuvant or other
from a single bulk into containers and processed together in auxiliary substance. It is treated to ensure its homogeneity
a single operation in such a manner as to ensure uniformity and is used for filling the containers of one or more final lots
and stability and to preyent contamination. A master seed lot (batches).
in liquid form is usually stored at or below - 70 oC. Finallot (Batch) A collection of c1osed, final containers
A freeze-dried master seed lot is stored at a temperature or other final dosage units that are expected to be
known to ensure stability. homogeneous and equivalent with respect to risk of
Working seed lot A culture of a micro-organism derived contamination during filling or preparation of the final
from the master seed lot and intended for use in production. product. The dosage units are filled, or otherwise prepared,
Working seed lots are distributed into containers and stored from the same final bulk vaccine, freeze-dried together (if
as described aboye for master seed lots. applicable) and c10sed in one continuous working session.
Cell-bank system (Cell-seed system) A system whereby They bear a distinctive number or code identifying the final
successive finallots (batches) of a product are manufactured lot (batch). Where a final bulk vaccine is filled and/or freeze-
by culture in cells derived from the same master cell bank dried on several separate sessions, there results a related set
(master cell seed) . A number of containers from the master of final lots (batches) that are usuaHy identified by the use of
cell bank (master cell seed) are used to prepare a working a common part in the distinctive number or code; these
ceH bank (working ceH seed). The ceH-bank system (cell-seed related final lots (batches) are sometimes referred to as sub-
system) is validated for the highest passage level achieved batches, sub-Iots or filling lots.
during routine production. Combined vaccine A multicomponent preparation
Master cell bank (Master cell seed) A culture of ceHs formulated so that different antigens are administered
distributed into containers in a single operation, processed simultaneously. The different antigenic components are
together and stored in such a manner as to ensure uniformity intended to protect against different strains or types of the
and stability and to prevent contamination. A master ceH same organism and/or different organisms. A combined
bank (master ceH seed) is usually stored at - 70 oC or vaccine may be supplied by the manufacturer either as a
lower. single liquid or freeze-dried preparation or as several
Working cell bank (Working cell seed) A culture of cells constituents with directions for admixture before use.
derived from the master ceH bank (master cell seed) and
intended for use in the preparation of production cell
cultures. The working cell bank (working cell seed) is B. Aluminium in Adsorbed Vaccines
distributed into containers, processed and stored as described
for thé master cell bank (master cell seed).
Homogenise the preparation to be examined and transfer a
Primary cell cultures Cultures of cells obtained by suitable quantity, presumed to contain 5 mg to 6 mg of
trypsination of a súitable tissue or organ. The cells are aluminium, to a 50 mL combustion flask. Add 1 mL of
essentially identical to those of the tissue of origin and are no suljuric acid R, 0.1 mL of nitric acid R and sorne glass beads.
more than 5 in vitra passages from the initial preparation Heat the solution until thick, white fumes are evolved .
from the animal tissue. If there is charring at this stage add a few more drops of
Celllines Cultures of cells that have a high capacity for nitric acid R and continue boiling until the colour disappears.
multiplication in viu·a. In diploid cell lines, the cells have Allow to cool for a few minutes, carefully add 10 mL of
essentially the same characteristics as those of the tissue of water R and boil until a c1ear solution is obtained. Allow to
origino In continuous cell lines, the cells are able to multiply cool, add 0.05 mL of methyl arange salunan R and neutralise
indefinitely in culture and may be obtained from healthy or with strang sodium hydraxide salution R (6.5 mL to 7 mL). If a
tumoral tissue. Sorne continuous celllines have oncogenic precipitate forms dissolve it by adding, dropwise, sufficient
potential under certain conditions. dilute sulfuric acid R. Transfer the solution to a 250 mL
Production cell culture A culture of cells intended for conical flask, rinsing the combustion flask with 25 mL of
use in production; it may be derived from one or more water R. Add 25.0 mL of 0.02 M sadium edetate, 10 mL of
containers of the working cell bank (working ceH seed) or it acetate buffer solutian pH 4.4 R and a few glass beads and boil
may be a primary cell culture. gently for 3 minoAdd 0.1 mL of pyridylaz anaphthal salutian R
and titrate the hot solution with 0.02 M copper sulfate until
2014 Appendix XV F V-A447
the colour changes to purplish-brown. Carry out a blank 9 giL solution of sodium ehloride R. Mix thoroughly.
titration omitting the vaccine. Centrifuge at 15 000 g for 60 mino Transfer the aqueous
1 mL of 0.02 M sodium edetate is equivalent to 0.5396 mg of phase to a 10 mL volumetric flask and dilute to volume
Al. with water R. If this procedure fails to separate the
aqueous phase, add 100 giL of polysorbate 20 R to the
sodium chloride solution and repeat the procedure but
centrifuge at 22 500 g.
C. Calcium in Adsorbed Vaccines (b) Add 1.0 mL of the vaccine to be examined to 1.0 mL
(Ph. Eur. method 2.5.14) of a 100 giL solution of sodium ehloride R and mix.
All solutions used jor this test must be prepared using water R. Centrifuge at 1000 g for 15 mino Transfer the aqueous
Determine the calcium by atomic emission spectrometry phase to a 10 mL volumetric flask and dilute to volume
(2.2.22, Method l). Homogenise the preparation to be with water R.
examined. To 1.0 mL add 0.2 mL of dilute hydroehlorie (c) Add 1.0 mL of the vaccine to be examined to 2.0 mL of
acid R and dilute to 3.0 mL with water R. Measure the a 100 gIL solution of sodium ehloride R and 3.0 mL of
absorbance at 620 nm. ehlorojorm R and mix. Centrifuge at 1000 g for 5 mino
Transfer the aqueous phase to a 10 mL volumetric flask
and dilute to volume with water R.
D. Free Formaldehyde
U se method A, unless otherwise prescribed. Method B is E. Phenol in Immunosera (Antisera) and
suitable for vaccines where sodium metabisulfite has been
used to neutralise excess formaldehyde.
Vaccines
METHODA Homogenise the preparation to be examined. Dilute an
For vaccines for human use, prepare a 1 in 10 dilution of the appropriate volume with water R so as to obtain a solution
vaccine to be examined. For bacterial toxoids for veterinary presumed to contain 15 Jlg of phenol per millilitre. Prepare a
use, prepare a 1 in 25 dilution of the vaccine to be examined. series of reference solutions with phenol R containing 5 Jlg,
To 1 mL of the dilution, add 4 mL of water R and 5 mL of 10 llg, 15 llg, 20 llg and 30 ~lg of phenol per millilitre
aeetylaeetone reagent R1. Place the tube in a water-bath at respectively. To 5 mL of the solution to be examined and to
40 oC for 40 mino Examine the tubes down their vertical 5 mL of each of the reference solutions respectively, add
axes. The solution is not more intensely coloured than a 5 mL of buffer solution pH 9. O R, 5 mL of aminopyrazolone
standard, prepared at the same time and in the same so/ution R and 5 mL of potassium jerrieyanide solution R. Allow
manner, using 1 mL of a dilution of jormaldehyde solution R to stand for 10 min and measure the intensity of colour at
containing 20 llg of formaldehyde (CH 2 0) per millilitre, 546 nm.
instead of the dilution of the vaccine to be examined. Plot the calibration curve and calculate the phenol content of
the preparation to be examined.
METHODB
Test solution Prepare a 1 in 200 dilution of the vaccine to
be examined with water R. If the vaccine is an emulsion,
prepare an equivalent dilution using the aqueous phase F. Neurovirulence
separated by a suitable procedure (se e below). If one of the
1. Test for neurovirulence oflive virus vaccines
methods described below is used for separation of the
aqueous phase, a 1 in 20 dilution of the latter is used. (Ph. Eur. method 2.6.18)
For each test, use not fewer than ten monkeys that are
Reference solutions Prepare solutions containing
seronegative for the virus to be tested. For each monkey,
0.25 giL, 0.50 gIL, 1.00 giL and 2.00 gIL of CH2 0 by
inject not more than 0.5 mL of the material to be examined
dilution of jormaldehyde solution R with water R. Prepare a 1
into the thalamic region of each hemisphere, unless otherwise
in 200 dilution of each solution with water R.
prescribed. The total amount of virus inoculated in each
To 0.5 mL of the test solution and of each of the reference monkey must be not les s than the amount contained in the
solutions in test-tubes, add 5.0 mL of a freshly prepared recommended single human dose of the vaccine. As a check
0.5 giL solution of methylbenzothiazolone hydrazone against the introduction of wild neurovirulent virus, keep a
hydroehloride R. Close the tubes, shake and allow to stand for group of not fewer than four control monkeys as cage-mates
60 mino Add 1 mL of jerrie ehloride-sulfamie aeid reagent R or in the immediate vicinity of the inoculated monkeys.
and allow to stand for 15 mino Measure the absorbance Observe the inoculated monkeys for 17 to 21 days for
(2.2.25) of the solutions at 628 nm. Calculate the content of symptoms of paralysis and other evidence of neurological
formaldehyde in the vaccine 10 be examined from the involvement; observe the control monkeys for the same
calibration curve established using the reference solutions. period plus 10 days. Animals that die within 48 h of injection
The test is invalid if the correlation coefficient (r) of the are considered to have died from non-specific causes and
calibration curve is les s than 0.97. may be replaced. The test is not valid if: more than
Emulsions If the vaccine to be examined is an emulsion, 20 per cent of the inoculated monkeys die from nonspecific
the aqueous phase is separated using a suitable procedure causes; serum samples taken from the control monkeys at the
and used for preparation of the test solution. The following time of inoculation of the test animal s and 10 days after the
procedures have been found suitable. latter are euthanised show evidence of infection by wild virus
(a) Add 1.0 mL of the vaccine to be examined to 1.0 mL of of the type to be tested or by measles virus. At the end of the
isopropyl myristate R and mix. Add 1.3 mL of 1 M observation period, carry out autopsy and histopathological
hydroehloric aeid, 2.0 mL of ehlorojorm R and 2.7 mL of a examinations of appropriate areas of the brain for evidence of
V -A448 Appendix XV F 2014
central nervous system involvement. The material complies (a) 12 sections representative of the whole of the lumbar
with the test if there is no unexpected c1inical or enlargement,
histopathological evidence of involvement of the central (b) 10 sections representative of the whole of the cervical
nervous system attributable to the inoculated virus. enlargement,
(c) 2 sections from the medulla oblongata,
2.Test for neurovirulence ofpoliomyelitis vaccine
(oral) (d) 1 section from the pons and cerebellum,
(e) 1 section from the midbrain,
Monkeys used in the neurovirulence test comply with the (f) 1 section from the left and the right of the thalamus,
requirements given in the monograph on Poliomyelitis vaccine (g) 1 section from the left and the right motor cerebral
oral (0215) and weigh not less than 1.5 kg. cortex.
The pathogenicity for Macaca or Cercopithecus monkeys is Scoring of virus activity For the evaluation of virus
tested in comparison with that of a reference virus activity in the hemisections of the spinal cord and brain-
preparation for neurovirulence testing by inoculation into the stem, a score system for the severity of lesions is used,
lumbar region of the central nervous system after sedation differentiating cellular infiltration and destruction of neurons
with a suitable substance, for example, ketamine as follows:
hydrochloride. A sample of serum taken before the injection l. Cellular infiltration only (the monkey is not counted as
shall be shown not to contain neutralising antibody at a positive),
dilution of 1:4 when tested against not more than 1000
2. Cellular infiltration with minimal neuronal damage,
CCID so of each of the three types of poliovirus .
Number of monkeys The vaccine and the appropriate 3. Cellular infiltration with extensive neuronal damage,
homotypic reference virus are tested concurrently in the 4. Massive neuronal damage with or without cellular
same grQUP of monkeys . Equal numbers of animals are infiltration.
inoculated with the vaccine to be examined and the reference The scores are recorded on a standard form l . A monkey with
preparation. The animals are allocated randomly to neuronal lesions in the sections but that shows no needle
treatrnent groups and cages and their identity is coded so tract is counted as positive. A monkey showing a needle tract
that the treatment received by each animal is concealed from in the sections, but no neuronallesions is not regarded as
the observers and the evaluators of the sections. The number positive. A section that shows damage from trauma but no
of monkeys inoculated is such that in the evaluation of both specific virus lesions is not inc1uded in the score.
the vaccine and the reference preparation not fewer than Severity scores are based on hemisection readings of the
eleven positive monkeys are inc1uded for type 1 and type 2 lumbar (L), cervical (C) and brain (B) histological sections.
virus and not fewer than eighteen positive monkeys for type The lesion score (LS) for each positive monkey is calculated
3 virus (positive monkeys are those that show specific as follows:
neuronal lesions of poliovirus in the central nervous system).
More than one batch of vaccine may be tested with the same Sumaf Sumaf Sumaf
homotypic reference. Monkeys from the same quarantine
group are used wherever possible, otherwise monkeys from
two groups are used and equal numbers from each group are
L scare
[ N"mb,,",
+
e scare
Numberaf
,[ B scare
Number af
hemisect lons hemisections hemisectiOns
treated with the vaccine and the reference preparation. If the
test is carried out on two working days, an equal number of
monkeys from each group are inoculated on each day with A mean lesion score is calculated for each group of positive
the vaccine and the homotypic reference preparation. monkeys.
Virus content The virus contents of the vaccine and the Evaluation The comparison of the virus activity in the
homotypic reference preparation are adjusted so as to be as vaccine and the reference preparation is based on the activity
near as possible equal and between 10 5 . 5 and 10 6 .5 in the lumbar enlargement of the cord and the degree of
CCID solO.l mL. spread of activity from this region to the cervical enlargement
Observation All monkeys are observed for 17 to 22 days and the brain. Acceptance or rejection is based on the total
for signs of poliomyelitis or other virus infection. Monkeys score of all the test animals . Individual animals showing
that survive the first 24 h but die before the 11 ID day after evidence of unusually high activity, either in the lumbar
inoculation are autopsied to determine whether poliomyelitis region or as the result of spread from this region, are also
was the cause of death. Animals that die from causes other taken into consideration in the final evaluation.
than poliomyelitis are exc1uded from the evaluation. Animals The monovalent bulk passes the test if the required number
that become moribund or are severely paralysed are of animals is positive and if none of the c1inical and
euthanised and autopsied. All animal s that survive until the histopathological examinations shows a significant difference
end of the observation period are autopsied. The test is not in pathogenicity between the vaccine virus and the reference
valid if more than 20 per cent of the animals show material. Criteria for acceptance are given below.
intercurrent infection during the observation periodo eriteria A suitable number of neurovirulence qualifying
Number of sections examined The lumbar cord, the tests (for example, four tests) is carried out on each reference
cervical cord, the lower and upper medulla oblongata, the vaccine (types 1,2 and 3) to provide data on the activity of
midbrain, the thalamus and the motor cortex of each such vaccines that will serve as the basis of the criteria for
monkey, as a minimum, are subjected to histological vaccines to be tested. The overall mean lesion score (M) for
examination. Sections are cut with a thickness of 15 11m and the replicate tests on each reference virus is calculated
stained with gallocyanin. The minimum number of sections
examined is as follows: 1A suüable fonn is shown in ¡he Requiremems for Poliomyelitis Vaccine
(Oral) (Requiremems for Biological Substances No. 7, World Health
Organization) .
2014 Appendix XV G V -A449
together with the pooled estimate of the within-test variance

(i) and the within-test deviation (s).
G. Composition of Polysaccharide
Validity criteria for the results of a test on a reference Vaccines
preparation are established on the basis of the cumulative (Ph . Eur. methods 2.5.16 to 2.5.23 and 2.5.31)
data from the qualifying tests. No generally applicable criteria
can be given; for laboratories with limited experience, the Protein
following empirical method for setting acceptable limits for (Ph. Eur. method 2.5.16)
the mean lesion score for the reference preparation (XreV
Test solution Use a volumetric flask with a suitable
may be helpful (see Table 2.6.19.-1):
volume for preparation of a solution containing about 5 mg
per millilitre of dry polysaccharide. Transfer the contents of
Table 2.6.19.-1 a container quantitatively to the flask and dilute to volume
Lower limit Upper limit with water R. Place 1 mL of rhe solution in a glass rube and
Types 1 and 2 M- s M+s
add 0.15 mL of a 400 gIL solution of trichloroacetic acid R .
Shake, allow to stand for 15 min, centrifuge for 10 min at
Type 3 M- s!2 M+s
5000 r/min and discard the supernatant. Add 0.4 mL of
0.1 M sodium hydroxide to the centrifugation residue.
If the mean lesion score for the vaccine to be tested is X,es, Reference solutions Dissolve 0.100 g of bovine albumin R
and el' e2and C 3are constants determined as described in 100 mL of 0. 1 M sodium hydroxide (stock solution
below, then: containing 1 g of protein per litre). Dilute 1 mL of the stock
solution to 20 mL with 0.1 M sodium hydroxide (working
the vaccine is not acceptable if:
dilution 1: 50 mg ofprotein per litre) . Dilute 1 mL ofrhe
stock solution ro 4 mL with 0.1 M sodium hydroxide (working
X test - Xref > Cl dilution 2: 250 mg of protein per litre). Place in 6 glass
tubes 0.10 mL, 0.20 mL and 0.40 mL ofworking dilution 1
the vaccine may be retested once if: and 0.15 mL, 0.20 mL and 0.25 mL ofworking dilution 2.
Make up the volume in each tube to 0.40 mL using 0. 1 M
C1 < X test - Xref < C2 sodium hydroxide.
Prepare a blank using 0.40 mL of 0. 1 M sodium hydroxide.
If the vaccine is retested, the means of the lesion scores for Add 2 mL of cupri-tartaric solution R3 to each tube, shake
the vaccine to be tested and the reference vaccine are and allow to stand for 10 min. Add to each rube 0.2 mL of a
recalculated. The vaccine is not acceptable if: mixture of equal volumes of phosphomolybdotungstic reagem R
and water R, prepared immediately before use. Stopper the
X (testl+ t est2 ) - X(refl+ref2) >C tubes, mix by inverting and allow to stand in the dark for
3
2 30 min. The blue colour is stable for 60 min. If necessary,
centrifuge to obrain clear solutions.
The constants el' e2 and e3 are calculated from the Measure the absorbance (2.2.25) of each solution at 760 nm
expressions: using the blank as the compensation liquido Draw a
calibration curve from the absorbances of the 6 reference
solutions and the corresponding protein contents and read
from the curve the content of protein in the test solution.
(2i2 Nuc1eic Acids

C 2 = 2.6y M (Ph. Eur. method 2.5.17)
Test solution Use a volumetric flask with a suitable
volume for preparation of a solution containing abour 5 mg
per millilitre of dry polysaccharide. Transfer rhe contents of
a container quantitatively to the flask and dilute to volume
N1 number of positive monkeys per vaccine test, with water R.
N2 number of positive monkeys in the two tests, Dilute the test solution if necessary to obtain an absorbance
2.3 normal deviate at the 1 per cent level, value suitable for the instrument used. Measure the
2.6 normal deviate at the 0.5 per cent level, absorbance (2.2.25) ar 260 nm using water Ras the
1.6 normal deviate at the 5 per cent leve!. compensation liquido
A neurovirulence test in which the mean lesion score for the The absorbance of a 1 giL solution of nucleic acid at 260 nm
reference (Xref) is not compatible with previous experience is is 20.
not used for assessing a test vaccine. If the test is valid, the
mean lesion score for the vaccine to be tested (X,es,) is Phosphorus
calculated and compared with that of the homotypic (Ph. Eur. method 2.5.18)
reference vaccine. Test solution Use a volumen'ic flask with a suitable
volume for preparation of a solution containing about 5 mg
per millilitre of dry polysaccharide. Transfer the contents of
a container quantitatively to the flask and dilute to volume
with water R. Dilute the solution so that the volume used in
the test (1 mL) contains about 6 ~lg of phosphorus. Transfer
1.0 mL of the solution to a 10 mL ignition tube.
V-A450 Appendix XV G 2014
Reference solutions Dissolve 0.2194 g of pOlassium 1 mole of acetylcholine chloride (181. 7 g) is equivalent to 1
dihydrogen phosphate R in 500 mL of water R to give a mole of O-acetyl (43.05 g).
solution containing the equivalent of 0.1 mg of phosphorus
per millilitre. Dilute 5.0 mL of the solution to 100.0 mL Hexosamines
with water R. Introduce 0.5 mL, 1.0 mL and 2.0 mL of the (Ph. Eur. method 2.5.20)
dilute solution into 3 ignition tubes . Test solution Use a volumetric fiask with a suitable
Prepare a blank solution using 2.0 mL of water R in an volume for preparation of a solution containing about 5 mg
ignition tube. per millilitre of dry polysaccharide. Transfer the contents of
To all the tubes add 0.2 mL of sulfuric acid R and heat in an a container quantitatively to the fiask and dilute to volume
oil bath at 120 oC for 1 h and then at 160 oC until white with water R. Dilute the solution so that the volumes used in
fumes appear (about 1 h) . Add 0.1 mL of perchloric acid R the test contain 125 ¡.¡g to 500 f1g of glucosamine
and heat at 160 oC until the solution is decolorised (about (hexosamine). Introduce 1.0 mL of the diluted solution into
90 min). Cool and add to each tube 4 mL of water R and a graduated tube.
4 mL of ammonium mo1ybdate reagent R. Heat in a water-bath Reference solutions Dissolve 60 mg of glucosamine
at 37 oC for 90 min and coo!. Adjust the volume to 10.0 mL hydrochloride R in 100 mL of water R (stock solution
with water R. The blue colour is stable for several hours. containing 0.500 g of glucosamine per litre). Introduce
Measure the absorbance (2.2.25) of each solution at 820 nm 0.25 mL, 0.50 mL, 0.75 mL, and 1.0 mL of the working
using the blank solution as the compensation liquido Draw a dilution into 4 graduated tubes.
calibration curve with the absorbances of the 3 reference Prepare a blank using 1 mL of water R .
solutions as a function of the quantity of phosphorus in the Make up the volume in each tube to 1 mL with water R.
solutions and read from the curve the quantity of phosphorus Add 1 mL of a solution of hydrochloric acid R (292 gIL) to
in the test solution. each tube. Stopper the tubes and place in a water-bath for
1 h. Cool to room temperature. Add to each tube 0.05 mL
O-AcetyI Groups of a 5 gIL solution of thymolphthalein R in alcohol R; add a
(Ph. Eur. method 2.5.19) solution of sodium hydroxide R (200 gIL) until a blue colour is
Test solution Use a volumetric fiask with a suitable obtained and then 1 M hydrochl011C acid until the solution is
volume for preparation of a solution containing about 5 mg colourless. Dilute the volume in each tube to 10 mL with
per millilitre of dry polysaccharide. Transfer the contents of water R (neutralised hydrolysates).
a container quantitatively to the fiask and dilute to volume In a second series of 10 mL graduated tubes, place 1 mL of
with water R. Dilute the solution so that the volumes used in each neutralised hydrolysate. Add 1 mL of acetylacetone
the test contain 30 ¡.¡g to 600 ¡.¡g of acetylcholine chloride reagent (a mixture, prepared irnrnediately before use, of
(O-acetyl). Introduce 0.3 mL, 0.5 mL and 1.0 mL in 1 volume of acetylacetone R and 50 volumes of a 53 giL
duplicate into 6 tubes (3 reaction solutions and 3 correction solution of anhydrous sodium carbonate R) to each tube.
solutions). Stopper the tubes and place in a water-bath at 90 oC for
Reference solutions Dissolve 0.150 g of acetylcholine 45 mino Cool to room temperature. Add to each tube
chloride R in 10 mL of water R (stock solution containing 2.5 mL of alcohol R and 1.0 mL of
15 g of acetylcholine chloride per litre) . Immediately before dimethylaminobenzaldehyde solution (immediately before use
use, dilute 1 mL of the stock solution to 50 mL with water R dissolve 0.8 g of dimethylaminobenzaldehyde R in 15 mL of
(working dilution 1: 300 f1g of acetylcholine chloride per alcohol R and add 15 mL of hydrochloric acid R ) and dilute
millilitre). Irnrnediately before use, dilute 1 mL of the stock the volume in each tube to 10 mL with alcohol R . Stopper
solution to 25 mL with water R (working dilution 2: 600 ¡.¡g the tubes, mix by inverting -and allow to stand in the dark for
of acetylcholine chloride per millilitre) . Introduce 0.1 mL 90 mino Measure the absorbance (2.2.25) of each solution at
and 0.4 mL of working dilution 1 in duplicate (reaction and 530 nm using the blank as the compensation liquido
correction solutions) in 4 tubes and 0.6 mL and 1.0 mL of Draw a calibration curve from the absorbances for the 4
working dilution 2 in duplicate (reaction and correction reference solutions and the corresponding content of
solutions) in another 4 tubes. hexosamine and read from the curve the quantity of
Prepare a blank using 1 mL of water R. hexosamine in the test solution.
Make up the volume in each tube to 1 mL with water R.
Add 1.0 mL of 4 M hydrochloric acid to each of the correction Methylpentoses
tubes and to the blank. Add 2.0 mL of alkaline hydroxylamine (Ph. Eur. method 2.5.21)
solution R to each tube. Allow the reaction to proceed for Test solution Use a volumetric fiask with a suitable
exactly 2 min and add 1.0 mL of 4 M hydrochloric acid to volume for preparation of a solution containing about 5 mg
each of the reaction tubes. Add 1.0 mL of a 100 giL solution per millilitre of dry polysaccharide. Transfer the contents of
of Jerric chloride R in 0.1 M hydrochloric acid to each tube, a container quantitatively to the fiask and dilute to volume
stopper the tubes and shake vigorously to remove bubbles . with water R . Dilute the solution so that the volumes used in
Measure the absorbance (2.2.25) of each solution at 540 nm the test contain 2 f1g to 20 ¡.¡g of rhamnose (methylpentoses) .
using the blank as the compensation liquido For each reaction Introduce 0.25 mL, 0.50 mL and 1.0 mL of the diluted
solution, subtract the absorbance of the corresponding solution into 3 tubes .
correction solution. Draw a calibration curve from the Reference solutions Dissolve 0.100 g of rhamnose R in
corrected absorbances for the 4 reference solutions and the 100 mL of water R (stock solution containing 1 g of
corresponding content of acetylcholine chloride and read methylpentose per litre) . Irnrnediately before use, dilute
from the curve the content of acetylcholine chloride in the 1 mL of the stock solution to 50 mL with water R (working
test solution for each volume tested. Calculate the mean of dilution: 20 mg of methylpentose per litre). Introduce
the 3 values. 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL and 1.0 mL ofthe
working dilution into 5 tubes.
2014 Appendix XV G V-A451
Prepare a blank using 1 mL of water R. 150 kPa. Refill the cell with water R each time the volume of
Make up the volume in each tube to 1 mL with water R. liquid in it has decreased to 1 mL and continue until
Place the tubes in iced water and add dropwise and with 200 mL has been filtered and the remaining volume in the
continuous stirring to each tube 4.5 mL of a cooled mixture cell is about 2 mL. Using a syringe, transfer this residual
of 1 volume of water R and 6 volumes of sulfuric acid R. liquid to a 10 mL volumetric flask. Wash the cell with 3
Warm the tubes to room temperature and place in a water- quantities, each of 2 mL, of water R, transfer the washings to
bath for a few minutes. Cool to room temperature. Add to the flask and dilute to 10.0 mL with water R (test solution).
each tube 0.10 mL of a 30 giL solution of cysteine In each of 2 test-tubes place 2.0 mL of the test solution.
hydrochloride R, prepared immediately before use. Shake and Reference solutions Use the reference solutions
allow to stand for 2 h. prescribed in the monograph.
Measure the absorbance (2.2.25) of each solution at 396 nm Prepare 2 series of 3 test-tubes, place in the tubes of each
and at 430 nm using the blank as compensation liquido series 0.5 mL, 1.0 mL and 1.5 mL respectively, of the
For each solution, calculate the difference between the reference solution corresponding to the type of vaccine to be
absorbance measured at 396 nm and that measured at examined and adjust the volume in each tube to 2.0 mL with
430 nm. Draw a calibration curve from the absorbance water R .
differences for the 5 reference solutions and the Prepare blank solutions using 2.0 mL of water R in each of 2
corresponding content of methylpentose and read from the test-tubes.
curve the quantity of methylpentose in the test solution for To all the tubes add 5.0 mL of resorcinol reagent R. Heat at
each volume tested. Calculate the mean of the 3 values. 105 oC for 15 min, cool in cold water and transfer the tubes
to a bath of iced water. To each tube add 5 mL of isoamyl
Uronic Acids
alcohol R and mix thoroughly. Place in the bath of iced water
(Ph. Eur. method 2.5.22) for 15 min. Centrifuge the tubes and keep them in the bath
Test solution Use a volumetric flask with a suitable of iced water until the examination by absorption
volume for preparation of a solution containing about 5 mg spectrophotometry. Measure the absorbance (2.2.25) of each
per millilitre of dry polysaccharide. Transfer the contents of supernatant solution at 580 nm and 450 nm using isoamyl
a container quantitatively to the flask and dilute to volume alcohol R as the compensation liquid.For each wavelength,
with water R. Dilute the solution so that the volumes used in calculate the absorbance as the mean of the values obtained
the test contain 4 ¡.tg to 40 ¡.tg of glucuronic acid (uronic with 2 identical solutions. Subtract the mean value for the
acids). Introduce 0.25 mL, 0.50 mL and 1.0 mL of the blank solution from the mean values obtained for the other
diluted solution into 3 tubes. solutions.
Reference solutions Dissolve 50 mg of sodium Draw a graph showing the difference between the
glucuronate R in 100 mL of water R (stock solution absorbances at 580 nm and 450 nm of the reference
containing 0.4 g of glucuronic acid per litre) . Immediately solutions as a function of the content of N-acetylneuraminic
before use, dilute 5 mL of the stock solution to 50 mL with acid and read from the graph the quantity of
water R (working dilution: 40 mg of glucuronic acid per N-acetylneuraminic acid (sialic acid) in the test solution.
litre). Introduce 0.10 mL, 0.25 mL, 0.50 mL, 0.75 mL, and
1.0 mL of the working dilution into 5 tubes. Ribose
Prepare a blank using 1 mL of water R. (Ph. Eur. method 2.5.31)
Make up the volume in each tube to 1 mL with water R. Test solution Use a volumetric flask with a suitable
Place the tubes in iced water and add dropwise and with volume for preparation of a solution containing about 5 mg
continuous stirring to each tube 5.0 mL of borate solution R. per millilitre of dry polysaccharide. Transfer the contents of
Stopper the tubes and place in a water-bath for 15 mino Cool a container quantitatively to the ftask and dilute to volume
to room temperature. Add 0.20 mL of a 1.25 gIL solution of with water R. Dilute the solution so that the volumes used in
carbazole R in ethanol R to each tube. Stopper the tubes and the test contain 2.5 ¡.tg to 25 ¡.tg of ribose. Introduce
place in a water-bath for 15 mino Cool to room temperature. 0.20 mL and 0.40 mL of the diluted solution into tubes in
Measure the absorbance (2.2.25) of each solution at 530 nm triplicate.
using the blank as the compensation liquido Reference solutions Dissolve 25 mg of ribose R in water R
Draw a calibration curve from the absorbances for the 5 and dilute to 100.0 mL with the same solvent (stock solution
reference solutions and the corresponding content of containing 0.25 gIL of ribose). Immediately before use,
glucuronic acid and read from the curve the quantity of dilute 1 mL ofthe stock solution to 10.0 mL with water R
glucuronic acid in the test solution for each volume tested. (working dilution: 25 mg/L of ribose). Introduce 0.10 mL,
Calculate the mean of the 3 values. 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL and 1.0 mL of the
working dilution into 6 tubes.
Sialic Acid Prepare a blank using 2 mL of water R.
(Ph. Eur. method 2.5.23) Make up the volume in each tube to 2 mL with water R.
Test solution Transfer quantitatively the contents of one Shake. Add 2 mL of a 0.5 gIL solution ofjem'c ehloride R in
or several containers to a volumetric flask of a suitable hydrochlorie acid R to each tube. Shake. Add 0.2 mL of a
volume that will give a solution with a known concentration 100 gIL solution of orcinol R in ethanol R. Place the tubes in
of about 250 ~lg per millilitre of polysaccharide and dilute to a water-bath for 20 min. Cool in iced water. Measure the
volume with water R. Using a syringe, transfer 4.0 mL of this absorbance (2.2.25) of each solution at 670 nm using the
solution to a 10 mL ultrafiltration cell suitable for the blank as the compensation liquido Draw a calibration curve
passage of molecules of relative molecular mas s less than from the absorbance readings for the 6 reference solutions
50 000. Rinse the syringe twice with water R and transfer the and the corresponding content of ribose and read from the
rinsings to the ultrafiltration cel!. Carry out the ultrafiltration, curve the quantity of ribose in the test solution for each
with constant stirring, under nitrogen R at a pressure of about volume tested. Calculate the mean of the 3 values.
V-A452 Appendix XV H 2014
procedure 10 be followed in establishing and maintaining an

H. Chicken Flocks Free from Specified SPF flock is shown diagrammatically in Table 5.2.2.-2.
Pathogens for the Production and In order to establish a new SPF flock, a series of tests must
be conducted on 3 generations of birds . All birds in the 1sr
Quality Control of Vaccines generation must be tested at least once before the age of
(Ph. Eur. method 5.2.2) 20 weeks for freedom from avian leucosis group-antigen and
Where specified, chickens, embryos or cell culrures used for tested by an enzyme immunoassay (EIA) or by virus
the production or quality control ofvaccines are derived from neutralisation (VN) for freedom of antibodies to avian
eggs produced by chicken flocks free from specified leucosis virus subtypes A, B and J. All birds must also be
pathogens (SPF). The SPF starus of a flock is ensured by tested for freedom from antibodies 10 the vertically-
means of the system described below. The list of micro- transmissible agents listed in Table 5.2.2-1. From the age of
organisms given is based on current knowledge and will be 8 weeks the flock is tested for freedom from Salmonella.
updated as necessary. Clinical examination is carried out on the flock from 8 weeks
A flock is defined as a group of birds sharing a common of age and the birds must not exhibit any signs of infectious
environment and having their own caretakers who have no disease. The test methods to be used for these tests are given
contact with non-SPF flocks . Once a flock is defined, no in the table and further guidance is also given in the section
non-SPF birds are added ro ir. below on routine testing of designated SPF flocks. From
Each flock is housed so as ro minimise the risk of 20 weeks of age, the flock is tested as described under
contamination. The facility in which the flock is housed must Routine testing of designated SPF flocks. All stages of this
not be sited near ro any non-SPF flocks of birds with the testing regime are also applied to the subsequent
exception of flocks that are in the process of being 2 generations, except the testing of every bird before lay for
established as SPF flocks and that are housed in facilities and vertically-transmissible agents. All test results must indicate
conditions appropriate ro SPF flocks. The SPF flock is freedom from pathogens in all 3 generations for the flock
housed within an isolaror or in a building with filtered air consisting of the 3rd generation to be designated as SPF.
under positive pressure. Appropriate mea sures are taken ro SPF embryos derived from another designated SPF flock
prevent entry of rodents, wild birds, insects and unauthorised contained within a separate facility on the same site may be
personnel. introduced. From 8 weeks of age, these replacement birds are
Personnel authorised to enter the facility must have no regarded as a flock and are tested in accordance with test
contact with other birds or with agents potentially capable of procedures described aboye.
infecting the flock. Ir is advisable for personnel 10 shower and
Initial testing requirements for subsequent
change c10thing or to wear protective c10thing before entering
generations derived from a designated SPF ftock
the controlled facility.
Where a replacement flock is derived exc1usively from a fully
Wherever possible, items taken into the facility are sterilised.
In particular it is recommended that the feed is suitably established SPF flock the new generation is tested prior to
being designated as SPF. In addition to the tests for
treated to avoid introduction of undesirable micro-organisms
Salmonella and moni1Oring of the general health and
and that water is at least of potable quality, for example from
performance of the flock, further specific testing from the age
a chlorinated supply. No medication is administered ro birds
~ of 8 weeks is required. Tests are performed on two
within the flock that might interfere with detection of any
disease. 5 per cent samples of the flock (minimum 10, maximum 200
birds) taken with an interval of at least 4 weeks between the
A permanent record is kept of the general health of the flock ages of 12-16 weeks and 16-20 weeks.
and any abnormality is investigated. Factors to be monitored
All samples are collected and tested individually. Blood
inc1ude morbidity, mortality, general physical condition, feed
consumption, daily egg production and egg quality, fertility samples for antibody tests and suitable samples for testing for
and hatchability. Records are maintained for a period of at leucosis antigen are collected. The test methods to be used
are as described under Routine testing of designated SPF
least 5 years. Details of any deviation from normal in these
flocks . Only when all tests have confirmed the absence of
performance parameters or detection of any infection are
infection may the new generation be designated as SPF.
notified to the users of the eggs as soon as practicable.
The tests or combination of tests described below must have Routine testing of designated SPF ftocks
suitable specificity and sensitivity with respect to relevant
General examination and necropsy
serotypes of the viruses . Samples for testing are taken at
random. Clinical examination is carried out at least once per week
throughout the life of the flock in order 10 verify that the
A positive result for chicken anaemia virus (CAV) does not
birds are free from fowl-pox virus and signs of any other
necessarily exc1ude use of material derived from the flock
infection. In the event of mortality exceeding 0.2 per cent per
but live vaccines for use in birds less than 7 days old shall be
week, necropsy is performed on all available carcasses to
produced using material from CAV-negative flocks.
verify that there is no sign of infection. Where appropriate,
Inactivated vaccines for use in birds less than 7 days old may
his1Opathological and/or microbiological/virological srudies are
be produced using material from flocks that have not been
performed to confirm diagnosis. Specific examination for
shown to be free from CAV, provided it has been
tuberculosis lesions is carried out and his1Ological samples
demonstrated that the inactivation process inactivates CAVo
from any suspected lesions are specifically stained to verify
Establishment of an SPF ftock freedom from Mycobactenum avium. Caecal contents of all
available carcasses are examined microbiologically for the
A designated SPF flock is derived from chickens shown 10 be presence of Salmonella spp. using the techniques described
free from vertically-transmissible agents listed in below. Where appropriate, cae cal samples from up to 5 birds
Table 5.2.2-1. This isachieved by testing of2 generations may be pooled.
prior to the designated SPF flock. A general scheme for the
2014 Appendix XV H V-A453
Table 5.2.2.-1
Agent Test Vertical Rapid/slow
to be used" transmission spread
Avian adenoviruses, group 1 AGP, EIA yes slow
Avian encephalomyelitis virus AGP, EIA yes rapid
Avian infectious bronchitis virus HI, EIA no rapid
Avian infectious laryngotracheitis virus VN, EIA no slow
Avian leucosis viruses E[A for virus, yes slow
VN, EIA for antibody
Avian nephritis virus [S no slow
Avian orthoreoviruses [S, EIA yes slow
Avian reticuloendotheliosis virus AGP, IS, E[A yes s[ow
Chicken anaemia virus IS, EIA, VN yes slow
Egg drop syndrome virus HI, ElA yes s[ow
[nfectious bursa[ disease virus Serotype 1: AGP, EIA, VN no rapid
Serotype 2: VN
Influenza A virus AGP, EIA, H[ no rapid
Marek's disease virus AGP no rapid
Newcastle disease virus HI,E[A no rapid
Turkey rhinotracheitis virus EIA no slow
Mycoplasma gallisepticum Agg and HI to confirm a yes slow
positive test,
EIA, HI
Mycoplasma synoviae Agg and HI to confirm a yes rapid
positive test,
EIA, HI
Salmonella pullorum Agg yes slow
Agg: agglutination HI: haemagglutination inhibition
AGP: agar gel precipitation; the technique is suitable where testing is carried IS: immunostaining
out weekly VN: virus neutralisation
EIA: enzyrne irnrnunoassay
"Subject to agreernent by the competent authority, other types of test rnay be used provided they are at least as sensitive as those indicated and of
appropriate specificity.
Cultural testing for Salmonella spp 4-week period, samples are taken from :> per cent (minimum
Cultural testing for Salmonella spp. is perforrned either by 10, maximum 200) of the flock. It is recommended that
testing samples of droppings or cloacal swabs or by testing of 1.25 per cent of the fiock is sampled each week since sorne
drag swabs. Where droppings or cloacal swabs are tested, a test methods for sorne agents must be conducted on a weekly
total of 60 samples within each 4-week period is tested basis. Table 5.2.2.-1 classifies the agents into those that
throughout the entire life of the flock. Tests may be spread rapidly through the fiock and those that spread slowly
perforrned on pools of up to 10 samples. Where drag swabs or may not infect the entire flock. For those agents listed as
are tested, a minimum of 2 drag swabs are tested during each slowly spreading, each sample is tested individualIy.
4-week period throughout the entire life of the flock. For those agents listed as rapidly spreading, at least
Detection of Salmonella spp . in these samples is perforrned by 20 per cent of the samples colIected in each 4-week period
pre-enrichment of the samples folIowed by culture using are tested individualIy or, where serum neutralisation or
Salmonella-selective media. EUSA tests are employed, alI of the samples may be tested
individualIy or by preparing pools of 5 samples, colIected at
Tests for avian leucosis antigen the same time.
Prior to the commencement of laying, cloacal swabs or blood Suitable methods to be used for detection of the agents are
samples (using buffy coat cultivation) are tested for the shown in Table 5.2.2.-1. Subject to agreement by the
presence of group-specific leucosis antigen. A total of
competent authority, other test methods may be used
5 per cent (minimum 10, maximum 200) of the flock is provided they are shown to be at least as sensitive as those
sampled during each 4-week periodoDuring lay, albumen indicated and of appropriate specificity.
samples from 5 per cent (minimum 10, maximum 200) of
the eggs are tested in each 4-week periodo Tests are Tests to be conducted at the end ofthe laying period
perforrned by EIA for group-specific antigen using methods
FolIowing the last egg colIection, final testing to confirrn the
that are capable of detecting antigen from subgroups A, B
absence of verticalIy-transmissible agents indicated in
and J.
Table 5.2.2.-1 is perforrned. After the last egg colIection, a
Testfor antibodies to other agents minimum of 5 per cent of the fiock (minimum 10, maximum
Tests for antibodies to alI agents listed in Table 5.2.2.-1 are 200) is retained for at least 4 weeks. Blood samples are
perforrned throughout the laying period of the fiock. In each colIected from every bird in the group during the 4-week
V-A454 Appendíx XV J 2014
Table 5.2.2-2. - Schematic description of the establishment and maintenance of SPF flocks
NEW STOCK Establish freedom from vertically·transmissible agents
Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
Test for Salmonella spp. and perform general clinical observation from 8 weeks of age
Carry out routine testing for specified agents from 20 weeks of age
2'" GENERATION Test all birds for avian leucosis antigen and antibodies prior to 20 weeks of age
3" GENERATION Test all birds for avian leucosis antigen and antibodies prior lo 20 weeks of age
DESIGNATE FLOCK AS SPF IF ALL TESTS ARE SATlSFACTORY
3" GENERATlON Carry out routine testing for specified agents from 20 weeks of age
Carry out post-lay testing for vertically·transmissible agents
SUBSEQUENT GENERATlONS Test two 5 per cent samples for avian leucosis antigen and for antibodies against specified
agents between 12 and 20 weeks of aile
Test for Salmonella spp. and perform general clinica! observation from 8 weeks of age
Carry out post-Iay testing for vertically·transmissib!e agents
period with at least 1.25 per cent of the birds (25 per cent of
the sample) being bled not earlier than 4 weeks after the final J. Cell Substrates tor the Production ot
egg collection. Serum samples are tested for vertically- Vaccines tor Human Use
transmissible agents (as defined by Table 5.2.2.-1) using the
methods indicated. Where sampling is performed on a weekly
This general chapter deals with diploid cell lines and
basis, at least 1.25 per cent of the birds (25 per cent of the
continuous cell lines used as cell substrates for the
sample) are tested each week during this periodo
production of vaccines for human use; specific issues relating
Altematively, within 4 weeks of the final egg collection blood
to vaccines prepared by recombinant DNA technology are
andJor other suitable sample materials are collected from at
covered by the monograph Products oi recombinant DNA
least 5 per cent of the fiock and tested for the presence of
technology (0784). Testing to be carried out at various stages
vertically-transmissible agents using validated nucleic acid
(ceH seed, master cell bank, working cell bank, ceHs at or
amplification techniques (2.6.21).
beyond the maximum population doubling level used for
Action to be taken in the event of detection of a production) is indicated in Table 5.2.3.-1. General provisions
specified agent for the use of celllines and test methods are given below.
Where primary cells or cells that have undergone a few
If evidence is found of contamination of the fiock by an passages without constitution of a ceH bank are used for
agent listed as slowly spreading in Table 5.2.2.-1, aH vaccine production, requirements are given in the individual
materials derived from the flock during the 4-week period monograph for the vaccine concemed.
immediately preceding the date on which the positive sample
was collected are considered unsatisfactory. Similarly, if Diploid celllines A diploid cell line has a high but finite
evidence is found of contamination of the fiock by an agent capacity for multiplication in vitro.
listed as rapidly spreading in Table 5.2.2.-1, all materials Continuous celllines A continuous ceH line has the
derived from the flock during the 2-week period irnmediately capacity to multiply indefinitely in vitro; the cells often have
preceding the date on which the positive sample was differences in karyotype compared to the original cells; they
collected are considered unsatisfactory. Any product may be obtained from healthy or tumoral tissue either from
manufactured with such materials, and for which the use of mammals or from insects.
SPF materials is required, is considered unsatisfactory and For injectable vaccines produced in continuous celllines, the
must be discarded; any quality control tests conducted using purification process is validated to demonstrate removal of
the materials are invalid. substrate-cell DNA to a level equivalent to not more than
Producers must notify users of aH eggs of the evidence of lOng per single human dose, unless otherwise prescribed.
contamination as soon as possible following the outbreak. Cell-bank system Production of vacdnes in diploid or
Any flock in which an outbreak of any specified agent is continuous ceH lines is based on a ceH-bank system. The in
confirmed may not be redesignated as an SPF fiock. vitro age of the ceHs is counted from the master ceH bank.
Any progeny derived from that flock during or after the Each working ceH bank is prepared from one or more
4-week period prior to the last negative sample being containers of the master cell bank. The use, identity and
collected may not be designated as SPF. inventory control of the containers is carefuHy documented.
Media and substances of human or animal origin The
composition of media used for isolation and aH subsequent
culture is recorded in detail, and if substances of human or
animal origin are used they must be free from extraneous
2014 Appendix XV J V-A455
agents (2.6.16) and must comply with the general chapter on Cel! lines that show the presence of retroviruses capable of
5.1.7. Viral safety. replication are not acceptable for production of vaccines.
If human albumin is used, it complies with the monograph Tumorigenicity For the preparation of live vaccines, the
Human albumin solution (0255). cellline must not be tumorigenic at any population doubling
If bovine serum is used, it complies with the monograph level used for vaccine production. Where a tumorigenic cel!
Bovine serum (2262). line is used for the production of other types of vaccine, the
purification process is validated to demonstrate that residual
Trypsin used for the preparation of cell cultures is examined
substrate-cell DNA is reduced to a level equivalent to not
by suitable methods and shown to be sterile and free from
more than lOng per single human dose of the vaccine,
mycoplasmas and viruses, notably pestiviruses, circoviruses
unless otherwise prescribed, and that substrate-cel! protein is
and parvoviruses.
reduced to an acceptable leve!.
Cell seed The data used to assess the suitability of the cell
A cell line that is known to have tumorigenic potential do es
seed comprises information, where available, on source,
not have to be tested further. If a cel! line is of unknown
history and characterisation.
tumorigenic potential, it is either regarded as being
Source 01 the cell seed For human celllines, the tumorigenic or it is tested for tumorigenicity using an in vivo
following information concerning the donor is recorded: test as described below and, optionally, an in vitro test if
ethnic and geographical origin, age, sex, general physiological additional information is needed. The tests are carried out
condition, tissue or organ used, results of any tests for using cells at or beyond the maximum population doubling
pathogens. leve! that will be used for vaccine production.
For animal celllines, the following information is recorded The MRC-5, WI-38 and FRhL-2 celllines are recognised as
concerning the source of the cells: species, strain, breeding being non-mmorigenic and further testing is not necessary.
conditions, geographical origin, age, sex, general physiological
Chromosoma! characterisation Diploid cell lines shall
condition, tissue or organ used, results of any tests for
be shown to be diploid. More extensive characterisation of a
pathogens.
diploid cell line by karyotype analysis is required if the
Cells of neural origin, such as neuroblastoma and P 12 cell removal of intact cells during processing after harvest has not
lines, may contain substances that concentrate agents of been validated. Samples from 4 passage levels evenly spaced
spongiform encephalopathies and such cells are not used for over the life-span of the cell line are examined. A minimum
vaccine production. of 200 cells in metaphase are examined for exact count of
History 01 the cell seed The following information is chromosomes and for frequency of hyperploidy, hypoploidy,
recorded: the method used to isolate the cell seed, culmre polyploidy, breaks and strucmral abnormalities.
methods, any other procedures used to establish the master The MRC-5, the WI-38 and the FRhL-2 cel!lines are
cell bank, notably any that might expose the cells to recognised as being diploid and well characterised; where
extraneous agents. they are not genetically modified, further characterisation is
Full information may not be available on the ingredients of not necessary.
media used in the past for cultivation of cells, for example on
the source of substances of animal origin; where justified and Test methods for cell cultures
authorised, cell banks already established using such media Morphology the morphology of the cells is adequately
may be used for vaccine production. described and documented.
Characterisatíon 01 the cell seed The fol!owing Identification Nucleic acid fingerprint analysis and a
properties are investigated: relevant selection of the following are used to establish the
(1) the identity of the cells (for example, isoenzymes, identity of the cells:
serology, nucleic acid fingerprinting); (1) biochemical characteristics (isoenzyme analysis);
(2) the growth characteristics ofthe cells and their (2) immunological characteristics (histocompatibility
morphological properties (optical and electron antigens);
microscopes); (3) cytogenetic markers.
(3) for diploid celllines, karyotype; Contaminating cells The nucleic acid fingerprint analysis
(4) for diploid cel!lines, the in vitro life span in terms of carried out for identification also serves to demonstrate
population doubling leve!. freedom from contaminating cells.
Cell substrate stability Suitable viability of the cel! line in Bacteria! and fungal contamination The master cel!
the intended storage conditions must be demonstrated. For a bank and each working cell bank comply with the test for
given product to be prepared in the cell line, it is necessary sterility (2.6.1), carried out using for each medium 10 mL of
to demonstrate that consistent production can be obtained sup"ernatant fluid from cell cultures . Carry out the test on
with cells at passage levels at the beginning and end of the 1 per cent of the containers, with a minimum of 2
intended span of use. containers.
Infectious extraneous agents Cell lines for vaccine Mycoplasmas (2.6.7) The master cell bank and each
production shall be free from infectious extraneous agents. working cell bank comply with the test for mycoplasmas.
Tests for extraneous agents are carried out as shown in Use one or more containers for the test.
Table 5.2.3.-1 using the methods described below. Spiroplasmas (insect celllines) The master cell bank
For cel!lines of insect origin, tests for specific viruses relevant and each working cell bank of insect cel!s are demonstrated
to the species of origin of the insect cells and for arboviruses to be free of spiroplasmas by a validated method approved
(arthropod - borne virus es) are applied. The panel of viruses by the competent authority. Use one or more containers for
tested is chosen according to the current state of scientific the test.
knowledge. Electron microscopy (insect celllines) The master cell
bank is examined by electron microscopy for the presence of
V-A456 Appendix XV J 2014
Table 5.2.3.-1 - Testing of celllines

Test Cell seed Master cell bank Working cell bank Cells at or beyond the
(MCB) (WCB) maximum population
doubling level used for
production
1. IDENTlTY AND PURITY
Morphology + + + +
Identilication : nudeic acid fingerprinting and a relevant

selection 01 the lollowing tests: biochemical (e.g. + + + +
isoenzymes), immunological (e.g. histocompatibility),
cytogenetic markers
+ (1)
Karyotype (diploid celllines) + +
Life span (diploid ceUlines) + +
2. EXTRANEOUS AGENTS
Bacterial and lungal contamination + +
Mycoplasmas + +
Spiroplasmas (insect celllines) + +

+ (3) + (3)
Electron microscopy (insect celllines)
Tests lor extraneous agents in cell cultures +
+(2)
Co-<:ultivation '
+(2)
Tests in animals and eggs
Specific tests lor possible contaminants depending on +(2)
the origin 01 the cells
Retroviruses
3. TUMORIGENICITY
Tumorigenicity
(1) The diploid character is established lor each working cell bank but using ceUs at or beyond the maximum population doubling level used lor production.
(2) Testing is carried out lor each working cell bank, but using ceUs at or beyond the maximum po pul atio n doubling level used lor production.
(3) Testing is carried out lor the master cell bank, but using cells at or beyond the maximum population doubling level used lor production.
(4) The MRC-5, WI-38 and FRhL-2 celllines are recognised as being non-tumorigenic and they need not be tested. Tests are not carried out on ceJllines
that are known or assumed to be tumorigenic, lor example CHO and BHK-21.
(5) Testing is carried out on the ceU seed, but using cells at or beyond the maximum population doubling level used lor production.
adventitious agents. Celllines are maintained at the haemagglutinating viruses, or on cells to detect
temperature routinely used for production and taken at or haemadsorbing viruses. The test for haemagglutinating
beyond the maximum population doubling leve!. In addition, viruses does not apply for arboviruses to be detected in
cell lines are maintained at temperatures aboye and below insect cells. The cells comply with the test if no evidence of
that routinely used for production and may also be subjected any extraneous agent is found.
to other treatments such as exposure to chemical stressors. Retroviruses Examine for the presence of retroviruses
The maintenance temperatures and treatments used are using:
agreed with the competent authority along with the number
(1) product-enhanced reverse transcriptase (PERT) assay
of sectioned cells to be examined.
(2.6.21) carried out for cell bank supernatants using cells
Test for extraneous agents in cell cultures The cells at or beyond the maximum population doubling level
comply with the test for haemadsorbing viruses and with the that will be used for production;
tests in ceH cultures for other extraneous agents given in
(2) transmission electron microscopy.
chapter 2.6.16 under Production cell culture: control cells.
If the cells are of simian origin, they are also inoculated into If test (1) and/or test (2) gives a positive result, test (3) is
rabbit kidney ceH cultures to test for herpesvirus B carried out:
(cercopithecid herpesvirus 1). (3) infectivity assays carried out on human cells with an
Co-cultivation For marnmalian and avian celllines, . endpoint PERT assay on the supernatant.
co-cultivate intact ami/or disrupted cells separately with other Since the sensitivity of PERT assays is very high,
ceH systems including human cells and simian cells. interpretation of a positive signal may be equivocal and a
For insect celllines, extracts of disrupted cells are incubated decision on the acceptability of a ceH substrate is based on all
with other cell systems, including human, simian, and at available data.
least 1 ceHline that is different from that used in production, Tests in anima1s lnject intramuscularly (or, for suckling
is permissible to insect viruses and allows detection of mice, subcutaneously) into each of the foHowing groups of
human arboviruses (for example BHK-21 ). Carry out animals 10 7 viable cells divided equally between the animals
examinations to detect possible morphological changes. in each group:
Carry out tests on the ceH culture fiuids to detect
2014 Appendix XV J V-A457
(1) 2 litters of suckling mice les s than 24 h old, comprising euthanised before the end of the test to avoid unnecessary
not fewer than 10 animals; suffering.
(2) 10 adult mice. At the end of the observation period alI animals, ineluding
Inject intracerebralIy into each of 10 adult mice 10 6 viable the reference group(s), are euthanised and examined for
ce lIs to detect the possible presence of lymphocytic gross and microscopic evidence of the proliferation of
choriomeningitis virus . inoculated cells at the site of injection and in other organs
(for example, lymph no des, lungs, kidneys and liver).
Observe the animals for at least 4 weeks. Investigate animals
that become sick or show any abnormality to establish the In alI test systems, the animals are observed and palpated at
cause of illness. The celIs comply with the test if no evidence regular intervals for the formation of nodules at the sites of
of any extraneous agent is found. The test is invalid if fewer injection. Any nodules formed are measured in
than 80 per cent of the animals in each group remain healthy 2 perpendicular directions, the measurements being recorded
and survive to the end of the observation periodo regularly to determine whether there is progressive growth of
the nodule. Animals showing nodules that begin to regress
Tests in eggs Using an inoculum of 10 6 viable cells per
during the period of observation are euthanised before the
egg, inoculate the celIs into the alIantoic cavity of ten 9- to
nodules are no longer palpable, and processed for histological
ll-day-old SPF embryonated hens' eggs (5.2.2) and into the
examination. Animals with progressively growing nodules are
yolk sac of ten 5- to 6-day-old SPF embryonated hens' eggs.
observed for 1-2 weeks. Among those without nodule
Incubate for not less than 5 days. Test the alIantoic fluids for
the presence of haemagglutinins using mammalian and avian formation, half are observed for 3 weeks and half for
12 weeks before they are euthanised and processed for
red blood cells; carry out the test at 5 ± 3 oC and 20-25 oC
histological examination. A necropsy is performed on each
and read the results after 30-60 mino The cells comply with
animal and ineludes examination for gross evidence of
the test if no evidence of any extraneous agent is found.
tumour formation at the site of injection and in other organs
The test is invalid if fewer than 80 per cent of the embryos
such as lymph no des, lungs, brain, spleen, kidneys and liver.
remain healthy and survive to the end of the observation
AlI tumour-like lesions and the site of injection are examined
periodo
histologically. In addition, since sorne cell lines may give rise
Specific tests for possib1e contaminants depending on to metastases without evidence of local tumour growth, any
the origin of the cells Tests for specific pathogens are detectable regionallymph no des and the lungs of all animals
carried out using nueleic acid amplification techniques . are examined histologically.
(NAT) (2.6.21) with or without prior amplification in cells .
The test is invalid if fewer than 9 of the 10 animals injected
Altematively, suitable serological techniques such as enzyme-
with the positive reference cells show progressively growing
linked immunosorbent assay, serum neutralisation and anti-
tumours.
body production tests in suitable permissive animals may be
used. For celllines of rodent origin, use either antibody Tests for tumorigenicity in vitro The folIowing test
production tests in mice, rats or hamsters or nueleic acid systems may be used:
amplification techniques (2.6.21) to detect species-specific (1) colony formation in soft agar gels;
viruses. Testing must take account of the origin and culture (2) production of invasive cell growth following inoculation
history of the cell lineo The tests are designed to detect into organ cultures;
potential contaminants, particularly those that are known to (3) study of transformation activity using, for example, the
infect latently the species of origin, for example simian virus 3T3 assay system for active oncogenes.
40 in rhesus monkeys or Flock house virus in insect cells.
Tests for tumorigenicity in vivo The test consists in
establishing a comparison between the continuous celI line
and a suitable positive control (for example, HeLa or Hep2
cells).
Animal systems thar have been shown to be suitable for this
test inelude :
(1) athymic mice (NulNu genotype);
(2) newbom mice, rats or hamsters that have been treated
with antithymocyte serum or globulin;
(3) thymectomised and irradiated mice that have been
reconstituted (r, B+) with bone marrow from healthy
mice.
Whichever animal system is selected, the cellline and the
reference celIs are injected ipto separate groups of 10 animals
each. In both cases, the inoculum for each animal is 10 7 cells
suspended in a volume of 0.2 mL, and thé injection may be
by either the intramuscular or the subcutaneous route.
Newbom animals are treated with 0.1 mL of antithymocyte
serum or globulin on days O, 2, 7 and 14 after birth.
A potent serum or globulin is one that suppresses the
immune mechanisms of growing animals to the extent that
the subsequent inoculum of 10 7 positive reference cells
regularly produces tumours and metastases. Severely affected
animals showing evident, progressively growing tumours are
V-A458 Appendix XVI 2014
has acquired a pink colour, the medium may be restored

Appendix XVI once by heating the containers in a water-bath or in free-
flowing steam until the pink colour disappears and cooling
quickly, taking care to prevent the introduction of non-sterile
A. Test for Sterility 1 air into the container. Do not use the medium for a longer
(Ph. Eur. method 2.6.1) storage period than has been validated.
The test is applied to substances, preparations or articles which, Fluid thioglycollate medium is to be incubated at 30-35 oc.
according to the Pharmacopoeia, are required to be sterile. For products containing a mercurial preservative that cannot
However, a satisfactory result on/y indicates that no contaminating be tested by the membrane-filtration method, fluid
micro-organism has been found in the sample examined in the thioglycollate medium incubated at 20-25 oC may be used
conditions of the test. instead of soya-bean casein digest medium provided that it
has been validated as described in growth promotion test.
Precautions against mÍcrobial contamination
Where prescribed or justified and authorised, the following
The test for sterility is carried out under aseptic conditions. altemative thioglycollate medium may be used. Prepare a
In order to achieve such conditions, the test environment has mixture having the same composition as that of the fluid
to be adapted to the way in which the sterility test is thioglycollate medium, but omitting the agar and the
performed. The precautions taken to avoid contamination are resazurin sodium solution, sterilise as directed aboye.
such that they do not affect any micro-organisms which are The pH after sterilisation is 7.1 ± 0.2. Heat in a water-bath
to be revealed in the test. The working conditions in which prior to use and incubate at 30-35 oC under anaerobic
the tests are performed are monitored regularly by conditions.
appropriate sampling of the working area and by carrying out
SOYA-BEAN CASEIN DIGEST MEDIUM
appropriate controls.
Pancreatic digest of casein 17.0 g
Culture media and incubation temperatures Papaic digest of soya-bean meal 3.0 g
Media for the test may be prepared as described below, or Sodium chloride 5.0 g
equivalent commercial media may be used provided that they Dipotassium hydrogen phosphate 2.5 g
comply with the growth promotion test. Glucose monohydrate/anhydrous 2.5 g/2 .3 g
Water R 1000 mL
The following culture media have been found to be suitable
pH after sterilisation 7.3 ± 0.2
for the test for sterility. Fluid thioglycollate medium is
primarily intended for the culture of anaerobic bacteria; Dissolve the solids in water R, warming slightly to effect
however, it will also detect aerobic bacteria. Soya-bean casein solution. Cool the solution to room temperature. Add 1 M
digest medium is suitable for the culture of both fungi and sodium hydroxide, if necessary, so that after sterilisation the
aerobic bacteria. solution will have a pH of 7.3 ± 0.2. Filter, if necessary, to
c1arity, distribute into suitable ves seis and sterilise using a
FLUID THIOGLYCOLLATE MEDIUM
validated process. Store at a remperature between 2 oC and
L-Cystine 0.5 g 25 oC in a sterile well-c1osed container, unless it is intended
Agar 0.75 g for immediate use. Do not use the medium for a longer
Sodium chloride 2.5 g storage period than has been validated .
Glucose monohydrate/anhydrous . 5.5 g/5.0 g
Yeast extract (water-soluble) Soya-bean casein digest medium is to be incubated at
5.0 g
20-25 oC.
Sodium thioglycollate or 0.5 g The media used comply with the following tests, carried out
Thioglycollic acid 0.3 mL before or in parallel with the test on the product to be
Resazurin sodium solution (1 gIL of resazurin l.0 mL examined.
sodium), freshly prepared Sterility Incubate portions of the media for 14 days.
Water R 1000 mL No growth of micro-organisms occurs.
pH after sterilisation 7.1 ± 0.2 Growth promotion test of aerobes, anaerobes and fungi
Mix the L-cystine, agar, sodium chloride, glucose, water- Test each batch of ready-prepared medium and each batch
soluble yeast extract and pancreatic digest of casein with the of medium prepared either from dehydrated medium or from
water R and heat until solution is effected. Dissolve the ingredients. Suitable strains of micro-organisms are indicated
sodium thioglycollate or thioglycollic acid in the solution and, in Table 2.6.l.-1.
if necessary, add 1 M sodium hydroxide so that, after Inoculate portions of fluid thioglycollate medium with a small
sterilisation, the solution will have a pH of 7.1 ± 0.2. number (not more than 100 CFU) of the following micro-
If filtration is necessary, heat th'e solution again without organisms, using a separate portion of medium for each of
boiling and filter while ho! through moistened filter paper. the following species of micro-organism: Clostridium
Add the resazurin sodium solution, mix and place the sporogenes, Pseudomonas aeruginosa, Staphylococcus aureus.
medium in suitable vessels which provide a ratio of surface to Inoculate portions of soya-bean casein digest medium with a
depth of medium such that not more than the upper half of small number (not more than 100 CFU) of the following
the medium has undergone a colour change indicative of micro-organisms, using a separate portion of medium for
oxygen uptake at the end of the incubation periodo Sterilise each of the following species of micro-organism: Aspergillus
using a validated process. If the medium is stored, store at a brasiliensis, Bacillus subtilis, Candida albicans. Incubate for not
temperature between 2 oC and 25 oC in a sterile, airtight more than 3 days in the case of bacteria and not more than
container. If more than the upper one-third of the medium 5 days in the case of fungi.
Seed lot culture maintenance techniques (seed-Iot systems)
1 This chapter has undergone pharmacopoeial hannonisation. See chapter
are used so that the viable micro-organisms used for
5.8. Pharmacopoeial harmonisation.Stmlity, Test/or
2014 Appendix XVI A V -A459
Table 2.6. L· L - Strains of the test micro-organisms suitable for use in the growth promotion test and the method su itab ility test
Aerobic bacteria
Slaphylococcus aureus ATCC 6538, ClP 4.83, NCTC 10788, NCIMB 9518, NBRC 13276
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134
Pseudomonas aeruginosa ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275
Anaerobic bacterium
Clostridium sporogenes ATCC 19404, CIP 79.3, NCTC 532, ATCC 11437, NBRC 14293
Fungi
Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594

Aspergil/us brasiliensis ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455
inoculation are not more than 5 passages removed from the controls are included, The technique of membrane filtration
original master seed-lot. is used whenever the nature of the product permits, that is,
The media are suitable if a clearly visible growth of the for filterable aqueous preparations, for a1coholic or oily
micro-organisms occurs. preparations and for preparations miscible with or soluble in
aqueous or oily solvents provided these solvents do not have
Method suitability test an antimicrobial effect in the conditions of the test.
Carry out a test as described below under Test for sterility of Membrane filtrarlon Use membrane filters having a
the product 10 be examined using exactly the same methods nominal pore size not greater than 0.45 flm whose
except for the fbllowing modifications. effectiveness 10 retain micro-organisms has been established.
Membrane filtrarlon After transferring the contents of Cellulose nitrate filters, for example, are used for aqueous,
the container or containers 10 be tested to the membrane oily and weakly a1coholic solutions and cellulose acetate
add an inoculum of a small number of viable micro- filters, for example, for strongly a1coholic solutions. SpecialIy
organisms (not more than 100 Cpu) to the final portion of adapted filters may be needed for certain products,
sterile diluent used to rinse the filter. e,g. for antibiotics.
Direct inocularlon After transferring the content of the The technique described below assumes that membranes
container or containers 10 be tested (for catgut and other about 50 mm in diameter will be used, If filters of a different
surgical sutures for veterinary use: strands) to the culture diameter are used the volumes of the dilutions and the
medium add an inoculum of a small number of viable washings should be adjusted accordingly. The filtration
micro-organisms (not more than 100 CFU) 10 the medium. apparatus and membrane are sterilised by appropriate means.
In both cases use the same micro-organisms as those The apparatus is designed so that the solution 10 be
described aboye under Growth promotion test of aerobes, examined can be introduced and filtered under aseptic
anaerobes and fungi. Perforrn a growth promotion test as a conditions; it permits the aseptic removal of the membrane
positive controL Incubate alI the containers containing for transfer to the medium or it is suitable for carrying out
medium for not more than 5 days. the incubation after adding the medium 10 the apparatus
itself.
If clearly visible growth of micro-organisms is obtained after
the incubation, visualIy comparable to that in the control Aqueous solutions If appropriate, transfer a smalI
vessel without product, either the product possesses no quantity of a suitable, sterile diluent such as a 1 gIL neutral
antimicrobial activity under the conditions of the test or such solution of meat or casein peptone pH 7.1 ± 0.2 onto the
activity has been satisfac10rily eliminated, The test for sterility membrane in the apparatus and filter. The diluent may
may then be carried out without further modification. contain suitable neutralising substances and/or appropriate
inactivating substances for example in the case of antibiotics .
If clearly visible growth is not obtained in the presence of the
product to be tested, visualIy comparable to that in the Transfer the contents of the container or containers to be
control vessels without product, the product possesses tested 10 the membrane or membranes, if necessary after
antimicrobial activity that has not been satisfactorily diluting 10 the volume used in the method suitability test
eliminated under the conditions of the test, Modify the with the chosen sterile diluent but in any case using not les s
conditions in order to eliminate the' antimicrobial activity and than the quantities of the product 10 be examined prescribed
repeat the method suitability test. in Table 2.6,1.-2. Filter immediately. If theproduct has
antimicrobial properties, wash the membrane not less than
This method suitability test is perforrned:
3 times by filtering through it each time the volume of the
a) when the test for sterility has to be carried out on a new chosen sterile diluent used in the method suitability test,
product; Do not exceed a washing cycle of 5 times 100 mL per filter,
b) whenever there is a change in the experimental even if during the method suitability test it has been
conditions of the test. demonstrated that such a cycle does not fully eliminate the
The method suitability test may be performed simultaneously antimicrobial activity. Transfer the whole membrane 10 the
with the test for sterility of the product to be examined. culture medium or cut it aseptically into 2 equal parts and
transfer one half to each of 2 suitable media. Use the same
Test for sterility ofthe product to be examined volume of each medium as in the method suitability test.
The test may be carried out using the technique of Altematively, transfer the medium onto the membrane in the
membrane filtration or by direct inoculation of the culture apparatus. Incubate the media for not less than 14 days.
media with the product 10 be examined. Appropriate negative
V-A460 Appendix XVI A 2014
Table 2 6.1.-2. - Minimum quantity to be used (OY each medium

Minimum quantity to be used for each medium unless other-
Quantity per container
wise justified and authorised
Liquids
- less than 1 mL The whole contents of each container
- 1·40 mL Half the contents of each container but not less than 1 mL
- greater than 40 mL and not greater than 100 mL 20 mL
- greater than 100 mL 10 per cent of the contents of the container but not less than 20 mL
Antibiolic liquids 1 mL
Insoluble preparations, creams and oinlmenls lo be suspended or emulsified Use the contents of each container to provide not less than 200 rng
Solids
- less than 50 mg The whole contents of each container
- 50 mg or more but less than 300 mg Half the contents of each container but not less than 50 rng
- 300 mg to 5 g 150mg
greater than 5 g 500 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30 cm long)
Soluble solids Use for each medium not less than the volume of the product it may be preferable to use a
quantity prescribed in Table 2.6.1.-2 of the product concentrated culture medium prepared in such a way that it
dissolved in a suitable solvent such as the solvent provided takes account of the subsequent dilution. Where appropriate,
with the preparation, water for injections, saline or a 1 gIL the concentrated medium may be added directly to the
neutral solution of meat or casein peptone and proceed with product in its container.
the test as described aboye for aqueous solutions using a Oily liquids U se media to which have been added a
membrane appropriate to the chosen solvento suitable emulsifying agent at a concentration shown to be
Oils and oily solutions Use for each medium not less appropriate in the method suitability test, for example
than the quantity of the product prescribed in polysorbate 80 at a concentration of 10 giL.
Table 2.6.1 .-2. Oils and oily solutions of sufficiently low Ointments and creams Prepare by diluting to about 1 in
viscosity may be filtered without dilution through a dry 10 by emulsifying with the chosen emulsifying agent in a
membrane. Viscous oils may be diluted as necessary with a suitable sterile diluent such as a 1 gIL neutral solution of
suitable sterile diluent such as isopropyl myristate shown not meat or casein peptone. Transfer the diluted product to a
to have antimicrobial activity in the conditíons of the test. .medium not containing an emulsifying agent.
Allow the oil to penetrate the membrane by its own weight
Incubate the inoculated media for not less than 14 days.
then filter, applying the pressure or suctíon gradually. Wash
Observe the cultures several times during the incubation
the membrane at least 3 times by filtering through it each
periodo Shake cultures containing oily products gently each
time about 100 mL of a suitable sterile solution such as
day. However when fluid thioglycollate medium is used for
1 giL neutral meat or casein peptone containing a suitable
the detection of anaerobic micro-organisms keep shaking or
emulsifying agent at a concentration shown to be appropriate
mixing to a minimum in order to maintain anaerobic
in the method suitability test, for example polysorbate 80 at
conditions.
a concentration of 10 gIL. Transfer the membrane or
membranes to the culture medium or media or vice versa as Catgut and other surgical sutures lor veterinary use
described aboye for aqueous solutions, and incubate at the Use for each medium not less than the quantities of the
. same temperatures and for the same times. product prescribed in Table 2.6.1.-2. Open the sealed
package using aseptic precautions and remove 3 sections of
Ointments and creams Use for each medium not les s
the strand for each culture medium. Carry out the test on 3
than me quantities of the product prescribed in
sections, each 30 cm long, cut off from the beginning, the
Table 2.6.1.-2. Ointments in a fatty base and emulsions of
centre and the end of the strand. Use whole strands from
the water-in-oil type may be diluted to 1 per cent in
freshly opened cassette packs. Transfer each section of the
isopropyl myristate as described aboye, by heating, if
strand to the selected medium. Use sufficient medium to
necessary, to not more than 40 oc. In exceptional cases it
cover adequately the material to be tested (20 mL to
may be necessary to heat to not more than 44 oc. Filter as
150 mL).
rapidly as possible and proceed as described aboye for oils
and oily solutions. Observation and interpretation of results
Direct inoculation of the culture medium Transfer the At intervals during the incubation period and at its
quantity of the preparation to be examined prescribed in conc1usion, examine the media for macroscopic evidence of
Table 2.6.1.-2 directly into the culture medium so that the microbial growth. If the material being tested renders the
volume of the product is not more than 10 per cent of the medium turbid so that the presence or absence of microbial
volume of the medium, unless otherwise prescribed. growth cannot be readily determined by visual examination,
If the product to be examined has antimicrobial activity, 14 days after the beginning of incubation transfer portions
carry out the test after neutralising this with a suitable (each not les s than 1 mL) of the medium to fresh ves seIs of
neutralising substance or by dilution in a sufficient quantity the same medium and then incubate the original and transfer
of culture medium. When it is necessary to use a large vessels for not less than 4 days.
2014 Appendix XVI B V-A461
Table 2.6.1.-3. - Minimum number of items fo be fesfed

Minimum number oí items to be tested íor each medium, unless
Number oí items in the batch'
otherwise justified and authorised"
Parenteral preparations
- Not more than 100 containers 10 per cent or 4 containers, whichever is the greater
- More than 100 but not more than 500 containers 10 containers
2 per cent or 20 containers (lO containers for large-volume parenterals)
- More than 500 containers
whichever is less
Ophthalmic and other non-injectable preparations
- Not more than 200 containers 5 per cent or 2 containers. whichever is the greater
- More than 200 containers 10 containers

- lf the product is presented in the form of single-dose containers. apply the
scheme shown above for preparations for parenteral administration
2 per cent or 5 packages whichever is the greater, up to a maximum total of
Catgut and other surgical sutures for veterinary use 20 packages
Bulk salid products
- Up to 4 containers Each container
- More than 4 containers but not more than 50 containers 20 per cent or 4 containers, whichever is the greater
- More than 50 containers 2 per cent or 10 containers, whichever is the greater
, If the batch size is not known, use the maximum number of items prescribed.
"If the contents of one container are enough to inoculate the 2 media, this column gives the number of containers needed for both the media together.
If no evidence of microbial growth is found, the product lO container is insufficient lO carry out the tests, the contents of
be examined complies with the test for sterility. If evidence of 2 or more containers are used to inoculate the different
microbial growth is found the product to be examined does media.
not comply with the test for sterility, unless it can be c1early
demonstrated that the test was invalid for causes unrelated to Minimum number of items to be tested
the product to be examined. The test may be considered The minimum number of items lO be tested in relation to the
invalid only if one or more of the following conditions are size of the batch is given in Table 2.6.1.-3.
fulfilled: Guidelines on the test for sterility are given in general chapter
a) the data of the microbiological monilOring of the sterility 5.1.9.
testing facility show' a fault;
b) a review of the testing procedure used during the test in
question reveals a fauIr; B. Microbiological Examination 01 Non-
c) microbial growth is found in the negative controls; sterile Products
d) after determination of the identity of the micro-
organisms isolated from the test, the growth of this 1. Test for Specified Micro-organisms 1
species or these species may be ascribed unequivocally lO (Ph. Eur. method 2.6.13)
fauIrs with respect lO the material andJor the technique 1 INTRODUCTlON
used in conducting the sterility test procedure.
The tests described hereafter will allow determination of the
If the test is dec1ared to be invalid it is repeated with the absence or limited occurrence of specified micro-organisms
same number of uriits as in the original test. that may be detected under the conditions described.
If no evidence of microbial growth is found in the repeat test The tests are designed primarily to determine whether a
the product examined complies with the test for sterility. substance or preparation complies with an established
If microbial growth 1S found in the repeat test the product specification for microbiological quality. When used for such
examined does not comply with the test for sterility. purposes, follow the instructions given below, inc1uding the
number of samples to be taken, and interpret the results as
Application of the test to parenteral preparations, stated below.
ophthalmic and other non-injectable preparations
Altemative microbiological procedures, inc1uding automated
required to comply with the test for sterility
methods, may be used, provided that their equivalence to the
When using the technique of membrane filtration, use, Pharmacopoeia method has been demonstrated.
whenever possible, the whole contents of the container, but
2 GENERAL PROCEDURES
not less than the quantities indicated in Table 2.6.1.-2,
diluting where necessary to about 100 mL with a suitable The preparation of samples is carried out as described in
sterile solution, such as 1 gIL neutral meat or casein peplOne. general chapter 2. 6.12.
When using the technique of direct inoculation of media, use If the product to be examined has antimicrobial activity, this
the quantities shown in Table 2.6 .1.-2, unless otherwise is insofar as possible removed or neutralised as described in
justified and authorised. The tests for bacterial and fungal general chapter 2.6.12.
sterility are carried out on the same sample of the product to
be examined. When the volume or the quantity in a single 1 This chapter has undergone pharmacopoeial harmonisazion. See Chapter
5.8. Parmacopoeial harmonisation.
V-A462 Appendix XVI B 2014
If surface-active substances are used for sample preparaúon, Verify suitable properties of relevant media as described in
their absence of toxicity for micro-organisms and their Table 2.6 .13.-1.
compatibility with inactivarors used must be demonstrated as Test for growth promoting properties, liquid media
described in general chapter 2.6.12.
Inoculate a portion of the appropriate medium with a smaIl
3 GROWTH-PROMOTlNG AND INHIBITORY PROPERTlES number (not more than 100 CFU) of the appropriate micro-
OF THE MEDIA, SUITABILITY OF THE TEST AND organismo Incubate at the specified temperature for not more
NEGATlVE CONTROLS
than the shortest period of time specified in the test. Clearly
The ability of the test to detect micro-organisms in the visible growth of the micro-organism comparable ro that
presence of the product to be tested must be established. previously obtained with a previously tested and approved
Suitability must be confirmed if a change in testing batch of medium occurS.
performance, or the product, which may afIect the outcome
of the test is introduced. Test for growth promoting properties, salid media
Perform the surface-spread method, inoculating each plate
3-1 Preparation oftest strains
with a smaIl number (not more than 100 CFU) of the
Use standardised stable suspensions of test strains or prepare appropriate micro-organismo Incubate at the specified
them as stated below. Seed lot culture maintenance temperature for not more than the shortest period of time
techniques (seed-Iot systems) are used so that the viable specified in the test. Growth of the micro-organism
micro-organisms used for inoculation are not more than comparable to that previously obtained with a previously
5 passages removed froID the original master seed-Iot. tested and approved batch of medium occurS.
3-1-1 Aerobic micro-organisms Grow each of the
Test for inhibitory properties, liquid or salid media
bacterial test strains separately in casein soya bean digest
broth or on casein soya bean digest agar at 30-35 oC for Inoculate the appropriate medium with at least 100 CFU of
18-24 h. Grow the test strain for Candida albicans separately the appropriate micro-organismo Incubate at the specified
on Sabouraud-dextrose agar or in Sabouraud-dextrose broth temperature for not less than the longest period of time
at 20-25 oC for 2-3 days. specified in the test. No growth of the test micro-organism
occurs.
- Staphylococcus aureus such as ATCC 6538, NCIMB 9518,
CIP 4.83 or NBRC 13276; Test for indicative properties
- Pseudomonas aeruginosa such as ATCC 9027, NCIMB Perform the surface-spread method, inoculating each plate
8626, CIP 82.118 or NBRC 13275; with a smaIl number (not more than 100 CFU) of the
- Escherichia coli such as ATCC 8739, NCIMB 8545, CIP appropriate micro-organismo Incubate at the specified
53.126 or NBRC 3972; temperature for a period of time within the range specified in
the test. Colonies are comparable in appearance and
- Salmonella enterica subsp. enterica serovar Typhimurium,
indication reactions to those previously obtained with a
such as ATCC 14028 or, as an altemative, Salmonella
previously tested and approved batch of medium.
entelica subsp . enterica serovar Abony such as NBRC
100797, NCTC 6017 or CIP 80.39; 3-4 Suitability of the test method
- Candida albicans such as ATCC 10231, NCPF 3179, IP For each product ro be tested, perform the sample
48.72 or NBRC 1594. preparation as described in the relevant paragraph in section
Use buffered sodium ch1oride-peprone solution pH 7.0 or 4. Add each test strain at the time of mixing, in the
phosphate buffer solution pH 7.2 to make test suspensions. prescribed growth medium. Inoculate the test strains
Use the suspensions within 2 h or within 24 h if stored at individuaIly. Use a number of micro-organisms equivalem ro
2-8 oc. not more than 100 CFU in the inoculated test preparation.
3-1-2 Clostridia Use Clostridium sporogenes such as ATCC Perform the test as described in the relevant paragraph in
11437 (NBRC 14293, NCIMB 12343, CIP 100651) or section 4 using the shortest incubation period prescribed.
ATCC 19404 (NCTC 5320r CIP 79.03) orNBRC 14293 . The specified micro-organisms must be detected with the
Grow the c10stridial test strain under anaerobic conditions in indication reactions as described in secúon 4.
reinforced medium for c10stridia at 30-35 oC for 24-48 h . Any antimicrobial activity of the product necessitates a
As an altemative ro preparing and then diluting down a fresh modificaúon of the test procedure (se e 4-5-3 of general
suspensión of vegetative ceIls of Cl. sporogenes, a stable spore chapter 2.6. 12) .
suspension is used for test inoculation. The stable spore If for a given product the antirnicrobial activity with respect
suspension may be maintained at 2-8 oC for a validated to a micro-organism for which testing is prescribed cannot be
periodo neutralised, then it is to be assumed that the inhibited micro-
3-2 Negative control organism will not be present in the producto
To verify testing conditions, a negative control is performed 4 TESTlNG OF PRODUCTS
using the chosen diluent in place of the test preparaúon. 4-1 Bile-tolerant gram-negative bacteria
There must be no growth of micro-organisms. A negative
4-1-1 Sample preparation and pre-incubation Prepare
control is also performed when testing the products as
a sample using a 1 in 10 dilution of not less than 1 g of the
described in section 4. A failed negaúve control requires an
product to be examined as described in. general chapter
investigation.
2.6.12, but using casein soya bean digest broth as the chosen
3-3 Growth promotion and inhibitory properties of the diluent, mix and incubate at 20-25 oC for a time sufficient to
media resuscitate the bacteria but not sufficient ro encourage
Test each batch of ready-prepared medium and each batch mulúplication of the organisms (usuaIly 2 h but not more
of medium prepared either from dehydrated medium or from than 5 h).
ingredients. 4-1-2 Test for absence Unless otherwise prescribed, use
the volume corresponding to 1 g of the product, as prepared
Table 2.6.13.-1 - Growth promoting, inhibitory and indicative properties of media

Medium Property Test strains
Test for bile-tolerant gram-negative Enterobacteria enrichment broth-Mossel Growth promoting E. coli
bacteria P. aeruginosa
Inhibitory S. aureus
Violet red bile glucose agar Growth promoting + indicative E. coli

P. aeruginosa
Test for Escherichia coli MacConkey broth Growth promoting E. coli
MacConkey agar Growth promoting + indicative E. coli
Test for Salmonella Rappaport Vassiliadis Salmonella Growth promoting Salmonella enterica subsp. en/erica
enrichment broth serovar Typhimurium or Salmonella
enterica subsp. en/erica serovar Abony
Xylose, lysine, deoxycholate agar Growth promoting + indicative Salmonella enterica subsp. en/erica
serovar Typhimurium or Salmonella
enterica subsp. en/erica serovar Abony
Test for Pseudomonas aeruginosa Cetrimide agar Growth promoting P. aeruginosa
Inhibitory E. colí
Test for Staphylococcus aureus Mannitol salt agar Growth promoting + indicative S. aureus
Inhibitory E. coli
Test for clostridia Reinforced medium for clostridia Growth promoting el. sporogenes
Columbia agar Growth promoting Cl. sporogenes
Test for Candida albicans Sabouraud dextrose broth Growth promoting C. albicans
Sabouraud dextrose agar Growth promoting + indicative C. albicans
in 4-1-1, to inoculate enterobacteria enrichment broth- 4-2 Escherichia coli

Mossel. Incubate at 30-35 oC for 24-48 h. Subculture on 4-2-1 Sample preparation and pre-incubation Prepare
plates of violet red bile glucose agar. Incubate at 30-35 oC a sample using a 1 in 10 dilution of not less than 1 g of the
for 18-24 h. product to be examined as described in general chapter
The product complies with the test if there is no growth of 2.6.12, and use 10 mL or the quantity corresponding to 1 g
colonies. or 1 mL to inoculate a suitable amount (determined as
4-1-3 Quantitative test described under 3-4) of casein soya bean digest broth, mix
4-1-3-1 Selection and suoculture Inoculate suitable and incubate at 30-35 oC for 18-24 h.
quantities of enterobacteria enrichment broth-Mossel with 4-2-2 Selection and sub culture Shake the container,
the preparation as described under 4-1-1 andlor dilutions of transfer 1 mL of casein soya bean digest broth to 100 mL of
it containing respectively 0.1 g, 0.01 .g and 0.001 g (or MacConkey broth and incubate at 42-44 oC for 24-48 h.
0.1 mL, 0.01 mL and 0.001 mL) ofthe product to be Subculture on a plate of MacConkey agar at 30-35 oC for
examined. Incubate at 30-35 oC for 24-48 h. Sub culture 18-72 h.
each of the cultures on a plate of violet red bile glucose agar. 4-2-3 Interpretation Growth of colonies indicates the
Incubate at 30-35 oC for 18-24 h . possible presence of E. eolio This is confirmed by
4-1-3-2Interpretation Growth of colonies constitutes a identification tests.
positive result. Note the smallest quantity of the product that The product complies with the test if no colonies are present
gives a positive result and the largest quantity that gives a or if the identification tests are negative.
negative resulto Determine from Table 2.6.13.-2 the probable 4-3 Salmonella
number of bacteria.
4-3-1 Sample preparation and pre-incubation Prepare
the product to be examined as described in general chapter
Table 2.6.13.-2 - lnterpretation of results 2.6.12, and use the quantity corresponding to not less than
Results for each quantity of product Probable 10 g or 10 mL to inoculate a suitable amount (determined
number of
0.1 g or 0.01 g or 0.001 g or bacteria per as described under 3-4) of casein soya bean digest broth,
0.1 mL 0.01 mL 0.001 mL gram or mix and incubate at 30-35 oC for 18-24 h.
millilitre of
.product 4-3-2 Selection and sub culture Transfer 0.1 mL of
+ + + > lO' casein soya bean digest broth tci 10 mL of Rappaport
+ + - Vassiliadis Salmonella enrichment broth and incubate at
< lO' and > 10 2
30-35 cC for 18-24 h. Sub culture on plates of xylose, Iysine,
+ - - < JO' and > 10 deoxycholate agar. Incubate at 30-35 oC for 18-48 h.
- - - < 10 4-3-3 Interpretation The possible presence of Salmonella
is indicated by the growth of well-developed, red colonies,
with or without black centres. This is confirmed by
identification tests.
The product complies with the test if colonies of the types The product complies with the test if colonies of the types
described are not present or if the confirmatory identification described are not present or if the confirmatory identification
tests are negative. tests are negative.
4-4 Pseudomonas aeruginosa 4-7 Gandida albicans
4-4-1 Sample preparation and pre-incubation Prepare 4-7 -1 Sample preparation and pre-incubation Prepare
a sample using a 1 in 10 dilution of not less than 1 g of the the product to be examined as described in general chapter
product to be examined as described in general chapter 2.6.12, and use 10 mL or the quantity corresponding to not
2.6.12, and use 10 mL or the quantity corresponding to 1 g les s than 1 g or 1 mL to inoculate 100 mL of Sabouraud-
or 1 mL to inoculate a suitable amount (determined as dextrose broth and mix. Incubate at 30-35 oC for 3-5 days.
described under 3-4) of casein soya bean digest broth and 4-7 -2 Selection and subculture Subculture on a plate of
mix. When testing transdermal patches, filter the volume of Sabouraud-dextrose agar and incubate at 30-35 oC for
sample corresponding to 1 patch of the preparation 24-48 h.
described under 4-5-1 in general chapter 2.6.12 through a
4-7 -3 Interpretation Growth of white colonies may
sterile filter membrane and place in 100 mL of casein soya
indicate the presence of C. albicans. This is confirmed by
bean digest broth, Incubate at 30-35 oC for 18-24 h.
identification tests.
4-4-2 Selection and sub culture Sub culture on a plate of
The product complies with the test if such colonies are not
cetrimide agar and incubate at 30-35 oC for 18-72 h .
present or if the confirmatory identification tests are negative.
4-4-3 Interpretation Growth of colonies indicates the
possible presence of P. aeruginosa. This is confirmed by
identification tests. The following section is given for infomzation.
The product complies with the test if colonies are not present 5 RECOMMENDED SOLUTIONS AND CULTURE MEDIA
or if the confirmatory identification tests are negative . . The following solutions and culture media have been found
to be satisfactory for the purposes for which they are
4-5 Staphylococcus aureus prescribed in the test for microbial contamination in the
4-5-1 Sample preparation and pre-incubation Prepare Pharmacopoeia . Other media may be used provided that
a sample using a 1 in 10 dilution of not less than 1 g of the their suitability can be demonstrated.
product to be examined as described in general chapter
Stock buffer solution Place 34 g of potassium dihydrogen
2.6.12, and use 10"mL or the quantity corresponding to 1 g
phosphate in a 1000 mL volumetric fiask, dissolve in
or 1 mL to inoculate a suitable amount (determined as
500 mL of purified water, adjust to pH 7.2 ± 0.2 with
described under 3-4) of casein soya bean digest broth and
sodium hydroxide, dilute to 1000.0 mL with purified water
mix. When testing transdermal patches, filter the volume of
and mix. Dispense into containers and sterilise. Store at
sample corresponding to 1 patch of the preparation 2-8 oc.
described under 4-5-1 in general chapter 2.6.12 through a
sterile filter membrane and place in 100 mL of casein soya Phosphate buffer solution pH 7.2 Prepare a mixture of
bean digest broth. Incubate at 30-35 oC for 18-24 h . stock buffer solution and purified water (1 :800 V/V) and
sterilise.
4-5-2 Selection and sub culture Subculture on a plate of
mannitol salt agar and incubate at 30-35 oC for 18-72 h. Buffered sodium chloride-peptone solution pH 7.0
4-5-3 Interpretation The possible presence of S. aureus is Potassium dihydrogen phosphate 3.6 g
indicated by the growth of yellow/white colonies surrounded Disodium hydrogen phosphate 7.2 g, equivalent to
by a yellow zone. This is confirmed by identification tests. dihydrate 0.067 M phosphate
The product complies with the test if colonies of the types Sodium chloride 4.3 g
described are not present or if the confirmatory identification Peptone (meat or casein) 1.0 g
tests are negative. Purified water 1000 mL
. 4-6 Glostridia Sterilise in an autoclave using a validated cycle.
4-6-1 Sample preparation and heat treatrnent Prepare Casein soya bean digest broth
a sample using a 1 in 10 dilution (with a minimum total
volume of 20 mL) of not less than 2 g or 2 mL of the
Papaic digest of soya bean 3.0 g
Sodium chloride 5.0 g
2.6. 12. Divide the sample into 2 portions of at least 10 mL.
Dipotassium hydrogen phosphate 2.5 g
Heat 1 portion at 80 oC for 10 min and cool rapidly. Do not
heat the other portion.
Purified water 1000 mL
4-6-2 Selection andsubculture Use 10 mL or the
quantity corresponding to 1 g or 1 mL of the product to be Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at
examined of both portions to inoculate suitable amounts 25 oc. Sterilise in an autoclave using a validated cycle.
(determined as described under 3-4) of reinforced medium Casein soya bean digest agar
for clostridia. Incubate under anaerobic conditions at
30-35 oC for 48 h. After incubation, make sub cultures from
Papaic digest of soya bean 5.0 g
each container on Columbia agar and incubate under
Sodium chIoride 5.0 g
anaerobic conditions at 30-35 oC for 48-72 h.
Agar 15.0 g
4-6-3 Interpretation The occurrence of anaerobic growth Purified water 1000 mL
of rods (with or without endospores) giving a negative
catalase reaction indica tes the presence of clostridia. This is Adjust the pH so that after sterilisation it is 7.3 ± 0.2 at
confirmed by identification tests. 25 oC. Sterilise in an autoclave using a validated cycle.
Sabouraud-dextrose agar MacConkey agar

Dextrose 40.0 g Pancreatic digest of gelatin 17.0 g
Mixture of peptic digest of animal tissue and 10.0 g Peptones (meat and casein) 3.0 g
pancreatic digest of casein (1: 1) Lactose monohydrate 10.0 g
Agar 15.0 g Sodium chloride 5.0 g
Purified water 1000 mL Bile salts 1.5 g
Agar 13.5 g
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at
25 oc. Sterilise in an autoclave using a validated cycle. Neutral red 30.0 mg
Crystal violet 1 mg
Potato dextrose agar Purified water 1000 mL
Infusion from pota toes 200 g Adjust the pH so that after sterilisation it is 7.1 ± 0.2 at
Dextrose 20 .0 g 25 cc. Boil for 1 min with constant shaking then sterilise in
Agar 15 .0 g an autoclave using a validated cycle.
Rappaport Vassiliadis Salmonella enrichment broth
Soya pep tone 4.5 g
25 oC. Sterilise in an autoclave using a validated cycle.
Magnesium chloride hexahydrate 29.0 g
Sabouraud-dextrose broth Sodium chloride 8.0 g
Dextrose 20.0 g Dipotassium phosphate 0.4 g
Mixture of peptic digest of animal tissue and 10.0 g Potassium dihydrogen phosphate 0.6 g
pancreatic digest of casein (1: 1) Malachite green 0.036 g
Purified water 1000 mL Purified water 1000 mL
Adjust the pH so that after sterilisation it is 5.6 ± 0.2 at Dissolve, warming gentIy. Sterilise in an autoclave using a
25 oC. Sterilise in an autoclave using a validated cycle. validated cycle, at a temperature not exceeding 115 oc.
The pH is to be 5.2 ± 0.2 at 25 oC after heating and
Enterobacteria enrichment broth-Mosse1 autoclaving.
Pancreatic digest of gelatin 10.0 g
Xylose, Iysine, deoxycholate agar
Dehydrated ox bile 20.0 g Xylose 3.5 g
Potassium dihydrogen phosphate 2.0 g L-Lysine 5.0 g
Disodium hydrogen phosphate dihydrate 8.0 g Lactose m onohydrate 7.5 g
Brilliant green 15 mg Sucrose 7.5 g
Purified water 1000 mL Sodium chloride 5.0 g
Yeast extract 3.0 g
Adjust the pH so that after heating it is 7.2 ± 0.2 at 25 oc. Phenol red 80 mg
Heat at 100 oC for 30 min and cool immediately. Agar 13.5 g
Violet red bile gIucose agar Sodium deoxycholate 2.5 g
Sodium thiosulfate 6.8 g
Yeast extract 3.0 g
Ferric ammonium citrate 0.8 g
Bile saIts 1.5 g
Sodium chloride 5.0 g Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 oC.
Glucose m onohydrate 10.0 g Heat to boiling, cool to 50 oC and pour into Petri dishes.
Agar 15.0 g Do not heat in an autoclave.
Neutral red 30 mg
Crystal violet 2 mg
Cetrirnide agar
Purified water 1000 mL Pancreatic digest of gelatin 20.0 g
Magnesium chloride 1.4 g
Adjust the pH so that after heating it is 7.4 ± 0.2 at 25 oc. Dipotassium sulfate 10.0 g
Heat to boiling; do not heat in an autoclave. Cetrimide 0.3 g
MacConkey broth Agar 13.6 g
Glycerol 10.0 mL
Lactose monohydrate 10.0 g
Dehydrated ox bile 5.0 g Heat to boiling for 1 min with shaking. Adjust the pH so that
Bromocresol purple lO mg after sterilisation it is 7.2 ± 0.2 at 25 cc. Sterilise in an
Purified water 1000 mL autoclave using a validated cycle.
25 oC. Sterilise in an au~oclave using a validated cycle.
Mannitol salt agar 2 GENERAL PRO CEDURES

Pancreatic digest of casein 5.0 g Carry out the determination under conditions designed to
Peptic digest of animal tissue 5.0 g avoid extrinsic microbial contamination of the product 10 be
Beef extract 1.0 g examined. The precautions taken to avoid contamination
D-Mannitol 10.0 g must be such that they do not affect any micro-organisms
Sodium chloride 75.0 g that are to be revealed in the test.
Agar 15.0 g If the product to be examined has antimicrobial activity, this
Phenol red 0.025 g is insofar as possible removed or neutralised. If inactivators
Purified water 1000 mL are used for this purpose, their efficacy and their absence of
toxicity for micro-organisms must be demonstrated.
Heat 10 boiling for 1 min with shaking. Adjust the pH so that
after sterilisation it is 7.4 ± 0.2 at 25 oC. Sterilise in an If surface-active substances are used for sample preparation,
autoclave using a validated cycle. their absence of toxicity for micro-organisms and their
compatibility with inactivators used must be demonstrated.
Reinforced medium for c10stridia
3 ENUMERATION METHODS
Beef extract 10.0 g Use the membrane tiltration method or the plate-count
Peptone 10.0 g methods, as preseribed. The most-probable-number (MPN)
Yeast extraet 3.0 g method is generally the least aceurate method for microbial
Soluble starch 1.0 g counts, however, for certain product groups with a very low
Glucose monohydrate 5.0 g bioburden, it may be the most appropriate method.
Cysteine hydrochloride 0.5 g
The choice of method is based on faetors such as the nature
of the produet and the required limit of micro-organisms .
Sodium acetate 3.0 g
The chosen method must allow testing of a sufficient sample
Agar 0.5 g
size to judge compliance with the specification.
The suitability of the method chosen must be established.
Hydrate the agar, dissolve by heating to boiling with
4 GROWTH PROMOTION TEST, SUITABILlTY OF THE
eontinuous stirring. If necessary, adjust the pH so that after COUNTING METHOD AND NEGATIVE CONTROLS
sterilisation it is 6.8 ± 0.2 at 25 oC. Sterilise in an autoclave
using a validated cycle. 4-1 General considerations
The ability of the test to detect micro-organisms in the
Columbia agar presence of product 10 be tested must be established.
Pancreatic digest of casein 10.0 g Suitability must be confirmed if a change in testing
Meat peptic digest 5.0 g performance, or the product, which may affect the outcome
Heart panereatic digest 3.0 g of the test is introduced.
Yeast extract 5.0 g
Maize starch 1.0 g 4-2 Preparation 01 test strains
Sodium chloride 5.0 g Use standardised stable suspensions of test strains or prepare
Agar, according to gelling power 10.0-15 .0 g them as stated below. Seed lot culture maintenance
Purified water 1000 mL teehniques (seed-Iot systems) are used so that the viable
micro-organisms used for inoculation are not more than
Hydrate the agar, dissolve by heating to boiling with
5 passages removed from the original master seed-lot. Grow
contiriuous stirring. If necessary, adjust the pH so that after
each of the bacterial and fungal test strains separately as
sterilisation it is 7.3 ± 0.2 at 25 oC. Sterilise in an autoclave
described in Table 2.6 .12.-1.
using a validated cycle. Allow to cool 10 45-50 oC; add,
where necessary, gentamicin sulfate corresponding to 20 mg Use buffered sodium chloride-peptone solution pH 7.0 or
of gentamicin base and pour into Petri dishes. phosphate buffer solution pH 7.2 10 make test suspensions;
to suspend A. brasiliensis spores, 0.05 per cent of polysorbate
2. Microbial Enumeration Tests l 80 may be added 10 the buffer. Use the suspensions within
(Ph. Eur. mechod 2.6.12) 2 h or within 24 h if stored at 2-8 oC . As an altemative 10
preparing and then diluting a fresh suspension of vegetative
I INTRODUCTION
cells of A. brasiliensis or B. subtilis, a stable spore suspension
The tests described hereafter will allow quantitative is prepared and then an appropriate volume of the spore
enumeration of mesophilic bacteria and fungi that may grow suspension is used for test inoculation. The stable spore
under aerobic conditions. suspension may be maintained at 2-8 oC for a validated
The tests are designed' primarily to determine whether a period of time.
substance or preparation complies with an established
specification for microbiological quality. When used for such 4-3 Negative control
purposes follow the instructions given below, including the To verify testing conditions, a negative control is performed
number of samples 10 be taken, and interpret the results as using the chosen diluent in place of the test preparation.
stated be1ow. There must be no growth of micro-organisms. A negative
control is also performed when testing the products as
The methods are not applicable to products containing viable
described in section 5. A failed negative control requires an
micro-organisms as active ingredients.
investigation.
Altemative microbiological procedures, including automated
methods, may be used, provided that their equivalence to the 4-4 Growth promotion 01 the media
Pharmacopoeia method has been demonstrated. Test each batch of ready-prepared medium and each batch
of medium, prepared either from dehydrated medium or
1 This chapter has undergone phannacopoeial harmonisation. See chapter from the ingredients described.
Table 2.6.12.-1. - Preparation and use oftest micro-organisms

Miero-organism Preparation of test Growth promotion Suitability of eounting method in the
strain presenee of the produet
Total aerobie mierobial Total yeasts and Total aerobie mierobial Total yeasts and
eount moulds eount eount moulds eount
Staphylococcus aureus Casein soya bean digest Casein soya bean digest Casein soya bean digest
su eh as: agar or easein soya agar and casein soya agar/MPN easein soya
bean digest broth bean digest broth bean digest broth
ATCC 6538
30.35 oc ,; 100 CFU ,; 100 CFU
NCIMB 9518
18·24 h 30.35 oC 30.35 oC
CIP 4.83
,; 3 days ,; 3 days
NBRC 13276
Pseudomonas Casein soya bean digest Casein soya bean digest Case in soya bean digest
aeruginosa agar or easein soya agar and case in soya agar/ MPN casein soya
sueh as: bean digest broth bean digest broth bean digest broth
ATCC 9027 . 30-35 oc ,; 100 CFU ,; 100 CFU
NCIMB 8626 18-24 h 30.35 oC 30-35 oc
CIP 82.118 ,; 3 days ,; 3 days
NBRC 13275
Bacillus subtilis Casein soya bean digest Casein soya bean digest Casein soya bean digest
sueh as: agar or easein soya agar and easein soya agar/MPN easein soya
ATCC 6633 bean digest broth bean digest broth bean digest broth
NCIMB 8054 30.35 oC ,; 100 CFU ,; 100 CFU
CIP 52.62 18·24 h 30-35 oC 30.35 oC
NBRC 3134 ,; 3 days ,; 3 days
Gandida albicans Sabouraud-dextrose Casein soya bean Sabouraud·dextrose Casein soya bean Sabouraud-dextrose
su eh as: agar or Sabouraud· digest agar agar digest agar agar
dextrose broth 20-25 oC ,; 100 CFU ,; 100 CFU ,; 100 CFU ,; 100 CFU
ATCC 10231
2·3 days 30.35 oC 20.25 oC 30.35 oC 20.25 oC
NCPF 3179
IP 48.72 ,; 5 days ,; 5 days ,; 5 days ,; 5 days
NBRC 1594 MPN: not applicable
Aspergillus brasiliensis Sabouraud-dextrose Casein soya bean Sabouraud-dextrose Casein soya bean Sabouraud-dextrose
sueh as: agar or potato-dextrose digest agar agar digest agar agar
agar ,; 100 CFU ,; 100 CFU ,; 100 CFU ,; 100 CFU
ATCC 16404
20.25 oC 30.35 oC 20.25 oC 30.35 oC 20.25 oC
IMI149007
5-7 days, or until good ,; 5 days ,; 5 days ,; 5 days ,; 5 days
IP 1431.83 sporulation is aehieved
NBRC 9455 MPN: not applieable
Inoculate portions/plates of casein soya bean digest broth and Non-fatty products insoluble in water Suspend the
casein soya bean digest agar with a small number (not 'more product to be examined (usually a 1 in 10 dilution is
than 100 CFU) of the micro-organisms indicated in prepared) in buffered sodium chloride-peptone solution
Table 2.6.12.-1, using a separate portion/plate of medium for pH 7.0, phosphate buffer solution pH 7.2 or casein soya
each. Inoculate plates of Sabouraud-dextrose agar with a bean digest broth. A surface-active agent such as 1 giL of
small number (not more than 100 CFU) of the micro- polysorbate 80 may be added to assist the suspension of
organisms indicated in Table 2.6.12.-1, using a separate plate poorly wettable substances. If necessary, adjust to pH 6-8.
of medium for each. Incubate in the conditions described in Further dilutions, where necessary, are prepared with the
Table 2.6.12.-1. same diluent.
For solid media, growth obtained ml,lst not differ by a factor Fatty products Dissolve in isopropyl myristate, sterilised
greater than 2 from the ca1culated value for a standardised by filtration or mix the product to be examined with the
inoculum. For a freshly prepared inoculum, growth of the minimum necessary quantity of sterile polysorbate 80 or
micro-organisms comparable to that previously obtained with another non-inhibitory sterile surface-active agent, heated if
a previously tested and approved batch of medium occurs. necessary to not more than 40 oC, or in exceptional cases to
Liquid media are suitable if c1early visible growth of the not more than 45 cC. Mix carefully and if necessary
micro-organisms comparable to that previously obtainedwith maintain the temperature in a water-bath. Add sufficient of
a previously tested and approved batch of medium occurS. the pre-warmed chosen diluent to make a 1 in 10 dilution of
4-5 Suitability of the counting method in the presence the original producto Mix carefully whilst maintaining the
ofproduct temperature for the shortest time necessary for the formation
of an emulsion. Further serial tenfold dilutions may be
4-5-1 Preparation ofthe sample The method for sample
prepared using the chosen diluent containing a suitable
preparation depends upon the physical characteristics of the
concentration of sterile polysorbate 80 or another non-
product to be tested. If none of the pro ce dures described
inhibitory sterile surface-active agent.
below can be demonstrated to be satisfactory, an altemative
procedure must be developed. Fluids or solids in aerosol form Aseptically transfer the
product into a membrane filter apparatus or a sterile
Water-soluble products Dissolve or dilute (usually a 1 in
container for further sampling. Use either the total contents
10 dilution is prepared) the product to be examined in
or a defined number of metered doses from each of the
buffered sodium chloride-peptone solution pH 7.0,
containers tested.
phosphate buffer solution pH 7.2 or casein soya bean digest
broth. If necessary, adjust to pH 6-8. Further dilutions, Transdermal patches Remove the protective cover sheets
where necessary, are prepared with the same diluent. ('releas e liners') of the transdermal patches and place them,
adhesive side upwards, on sterile glass or plastic trays. Cover
the adhesive surface with a sterile porous material, for organismo However, it is possible that the product only
example sterile gauze, to prevent the patches from sticking inhibits sorne of the micro-organisms specified herein, but
together, and transfer the patches to a suitable volume of the does not inhibit others not included amongst the test strains
chosen diluent containing inactivators such as polysorbate 80 or for which the latter are not representative. Then, perform
and/or lecithin. Shake the preparation vigorously for at least the test with the highest dilution factor compatible with
30 min. microbial growth and the specific acceptance criterion.
4-5-2 Inoculation and dilution Add to the sample 4-5-4 Recovery of micro-organism in the presence of
prepared as described aboye (4-5-1) and to a control (with product For each of the micro-organisms listed, separate
no test material included) a sufficient volume of the tests are performed. Only micro-organisms of the added test
microbial suspension to obtain an inoculum of not more strain are counted.
than 100 CFU. 'The volume ofthe suspension ofthe 4-5-4-1 Membrane jiltration Use membrane filters
inoculum should not exceed 1 per cent of the volume of having a nominal pore size not greater than 0.45 ~m.
diluted product. The type of filter material is chosen such that the bacteria-
To demonstrate acceptable microbial recovery from the retaining efficiency is not affected by the components of the
product, the lowest possible dilution factor of the prepared sample to be investigated. For each of the micro-organisms
sample must be used for the test. Where this is not possible listed, one membrane filter is used.
due to antimicrobial activity or poor solubility, further Transfer a suitable amount of the sample prepared as
appropriate protocols must be developed. If inhibition of described under 4-5-1 to 4-5-3 (preferably representing 1 g
growth by the sample cannot otherwise be avoided, the of the product, or less if large numbers of CFU are expected)
aliquot of the microbial suspension may be added after to the membrane filter, filter immediately and rinse the
neutralisation, dilution or filtration . membrane filter with an appropriate volume of diluent.
4-5-3 Neutralisation/removal of antimicrobial activity For the determination of total aerobic microbial count
The number of micro-organisms recovered from the (TAMC), transfer the membrane filter to the surface of
prepared sample diluted as described in 4-5-2 and incubated casein soya bean digest agar. For the determination of total
folIowing the procedure described in 4-5-4, is compared to combined yeasts/moulds count (TYMC), transfer the
the number of micro-organisms recovered from the control membrane to the surface of Sabouraud-dextrose agar.
preparation. . Incubate the plates as indicated in Table 2.6 .12.-1. Perform
If growth is inhibited (reduction by a factor greater than 2), the counting.
then modify the procedure for the particular enumeration test 4-5-4-2 Plate-count methods Perform plate-count
to ensure the validity of the results. Modification of the methods at least in duplicate for each medium and use the
procedure may include, for example, (1) an increase in the mean count of the resulto
volume of the diluent or culture medium, (2) incorporation 4-5-4-2-1 Pour-plate method
of specific or general neutralising agents into the diluent, (3)
membrane filtration, or (4) a combination of the aboye For Petri dishes 9 cm in diameter, add to the dish 1 mL of
measures. the sample prepared as described under 4-5-1 to 4-5-3 and
15-20 mL of casein soya bean digest agar or Sabouraud-
Neutralising agents Neutralising agents may be used to dextrose agar, both media being at not more than 45 oC.
neutralise the activity of antimicrobial agents
If larger Petri dishes are used, the amount of agar medium is
(Table 2.6 .12.-2). They may be added to the chosen diluent increased accordingly. For each of the micro-organisms listed
or the medium preferably before sterilisation. If used, their
in Table 2.6.12.-1, at least 2 Petri dishes are used . Incubate
efficacy and their absence of toxicity for micro-organisms
the plates as indicated in Table 2.6.12.-1. Take the
must be demonstrated by carrying out a blank with
arithmetic mean of the counts per medium and calcula te the
neutraliser and without product.
number of CFU in the original inoculum.
4-5-4-2-2 Surface-spread method
TabIe 2.6.12.-2. - Common neutralising agents for interfering
substances For Petri dishes 9 cm in diameter, add 15-20 mL of casein
soya bean digest agar or Sabouraud-dextrose agar at about
Interfering substance Potential neutralising 45 oC to each Petri dish and aIlow to solidify. If larger Petri
method
dishes are used, the volume of the agar is increased
Glutaraldehyde, mercurials Sodium hydrogensulfite
(sodium bisulfite) accordingly. Dry the plates, for example in a laminar-air-fiow
Phenolics, alcohol, aldehydes, sorbate Dilution cabinet or an incubator. For each of the micro-organisms
listed in Table 2.6.12.-1, at least 2 Petri dishes are used.
A1dehydes Glycine
Spread a measured volume of not less than 0.1 mL of the
Quaternary Ammonium Compounds Lecithin sample prepared as described under 4-5-1 to 4-5-3 over the
(QACs), parahydroxybenzoates (parabens),
bis·biguanides surface of the medium. Incubate and count as prescribed
QACs, iodine, parabens
under 4-5-4-2-1
Polysorbate
4-5-4-3 Most-probable-number (MPN) method The
Mercurials Thioglycollate
precision and accuracy of the MPN method is less than that
Mercurials, halogens, aldehydes Thiosulfate of the membrane filtration method or the plate-count
EDTA (edetate) Mg" or Ca" ions method. Unreliable results are obtained particularIy for the
enumeration of moulds. For these reasons the MPN method
is reserved for the enumeration of TAMC in situations
If no suitable neutralising method can be found, it can be where no other method is available. If the use of the method
assumed that the failure to isolate the inoculated organism is is justified, proceed as folIows.
attributable to the microbicidal activity of the product. This Prepare a series of at least 3 serial tenfold dilutions of the
information serves to indicate that the product is not likely to product as described under 4-5-1 to 4-5-3. From each level
be contaminated with the given species of the micro-
of dilution, 3 aliquots of 1 g or 1 mL are used to inoculate 3 Table 2.6.12.-3. - Most-probable-number values of

tubes with 9-10 mL of casein soya bean digest broth. micro-organisms
If necessary, a surface-active agent such as polysorbate 80 or Observed combinations of numbers of
an inactivator of antimicrobial agents may be added to the tubes showing growth in each se! MPN per
95 per cen!
Number oí grams or millilitres of gram or per
medium. Thus, if 3 levels of dilution are prepared, 9 tubes produc! per tube millilitre oí
confidence
are inoculated. Iimits
product
0.1 0.01 0.001
Incubate all tubes at 30-35 oC for not more than 3 days.
If reading of the results is difficult or uncertain owing to the O O O <3 0·9.4
nature of the product to be examined, subculture in the same O O 1 3 0.1-9.5
broth, or in casein soya bean digest agar, for 1-2 days at the
O 1 O 3 0.1-10
same temperature and use these n:sults. Detertnine the most
probable number of micro-organisms per gram or millilitre of O 1 1 6.1 1.2-17
the product to be examined from Table 2.6.12.-3. O 2 O 6.2 1.2·17
4-6 Results and interpretation O 3 O 9.4 3.5-35
When verifying the suitability of the membrane filtration 1 O O 3.6 0.2·17
method or the plate-count method, a mean count of any of
the test organisms not differing by a factor greater than 2 1 O 1 7.2 1.2·17
from the value of the control defined in 4-5-2 in the absence 1 O 2 11 4-35
of the product must be obtained. When verifying the
1 1 O 7.4 1.3·20
suitability of the MPN method the calculated value from the
inoculum must be within 95 per cent confidence limits of the 1 1 1 11 4-35
results obtained with the control. 1 2 O 11 4·35
If the aboye criteria cannot be met for one or more of the 1 2 1 15 5·38
organisms tested with any of the described methods, the
1 3 O 16 5·38
method and test conditions that come closest to the criteria
are used to test the product. 2 O O 9.2 1.5·35
5 TESTING OF PRODUCTS 2 O 1 14 4·35
5-1 Amount usedfor the test ' 2 O 2 20 5·38

Unless otherwise prescribed, use 10 g or 10 mL of the 2 1 O 15 4·38
product to be examined taken with the precautions referred
2 1 1 20 5·38
to aboye . For fiuids or solids in aerosol fortn, sample 10
containers. For transdertnal patches, sample 10 patches. 2 1 2 27 9·94
The amount to be tested may be reduced for active 2 2 O 21 5-40

substances that will be formulated in the following 2 2 1 28 9-94
conditions: the amount per dosage unit (e.g. tablet, capsule,
injection) is les s than or equal to 1 mg or the amount per 2 2 2 35 9·94
gram or millilitre (for preparations not presented in dose 2 3 O 29 9·94
units) is les s than 1 mg. In these cases, the amount to be 9·94
2 3 1 36
tested is not less than the amount present in 10 dosage units
or 10 g or 10 mL of the product. 3 O O 23 5·94
For materials used as active substances where sample 3 O 1 38 9·104

quantity is limited or batch size is extremely small (i.e. less 3 O 2 64 16-181
than 1000 mL or 1000 g), the amount tested shall be
3 1 O 43 9·181
1 per cent of the batch unless a les ser amount is prescribed
or justified and authorised. 3 1 1 75 17·199
For products where the total number of entities in a batch is 3 1 2 120 30-360
less than 200 (e.g. samples used in clinical trials), the sample
3 1 3 160 30-380
size may be reduced to 2 units, or 1 unit if the size is less
than 100. 3 2 O 93 18-360
Select the sample(s) at random from the bulk material or 3 2 1 150 30-380
from the available containers of the preparation. To obtain 3 2 2 210 30-400
the required quantity, mix the contents of a sufficient
3 2 3 290 90-990
number of containers to provide the sample.
3 3 O 240 40-990
5-2 Examination of the product
3 3 1 460 90·1980
5-2-1 Membrane filtration 3 3 2 1100 200-4000
Use a filtration apparatus designed to allow the transfer of
3 3 3 > 1100
the filter to the medium. Prepare the sample using a method
that has been shown suitable as described in section 4 and
transfer the appropriate amount to each of 2 membrane For the detertnination of TAMC, transfer one of the
filters and filter immediately. Wash each filter following the membrane filters to the surface of casein soya bean digest
procedure shown to be suitable. agar. For the detertnination of TYMC, transfer the other
membrane to the surface of Sabouraud-dextrose agar.
Incubate the plate of casein soya bean digest agar at 3. Test for Absence of Mycoplasmas
30-35 oC for 3-5 days and the plate of Sabouraud-dextrose (Ph . Eur. method 2.6.7 as applied to vaccines for human use)
agar at 20-25 cC for 5-7 days. Calculate the number of CFU Where the test for mycoplasmas is prescribed for a master
per gram or per millilitre of product. cell bank, for a working cell bank, for a virus seed lot or for
When examining transdermal patches, filter 10 per cent of control cells, both the culture method and the indicator cell
the volume of the preparation described under 4-5-1 culture method are used. Where the test for mycoplasmas is
separately through each of 2 sterile filter membranes. prescribed for a virus harvest, for a bulk vaccine or for the
Transfer one membrane to casein soya bean eligest agar for finallot (batch), the culture method is used. The indicator
T AMC and the other membrane to Sabouraud-dextrose agar cell culture method may also be used, where necessary, for
for TYMe. screening of media.
NucIeic acid amplification techniques (NAT) may be used as
5-2-2 Plate-count methods
an alternative to one or both of the other methods after
5-2-2-1 Pour-plate method suitable validation.
Prepare the sample using a method that has been shown to
be suitable as described in section 4. Prepare for each Culture method
medium at least 2 Petri dishes for each level of dilution. CHOICE OF CULTURE MEDIA
Incubate the pi ates of casein soya bean digest agar at The test is carried out using a sufficient number of both solid
30-35 oC for 3-5 days and the plates of Sabouraud-dextrose and liquid media to ensure growth in the chosen incubation
agar at 20-25 oC for 5-7 days. Select the pi ates conditions of small numbers of mycoplasmas that may be
corresponding to a given dilution and showing the highest present in the product to be examined. Liquid media must
number of colonies less than 250 for TAMC and 50 for contain phenol red. The range of media chosen is shown to
TYMC . Take the arithmetic mean per culture medium of have satisfactory nutritive properties for at least the micro-
the counts and calculate the number of CFU per gram or per organisms shown below. The nutritive properties of each new
millilitre of product. batch of medium are verified for the appropriate micro-
5-2-2-2 Surlace-spread method organisms in the listo When testing for mycoplasmas in the
Prepare the sample using a method that has been shown to product to be examined, at least 1 of the following species
be suitable as described in section 4. Prepare at least 2 Petri will be incIuded as a positive control:
dishes for each medium and each level of dilution. - Acholeplasma laidlawii (vaccines for human and veterinary
For incubation and calculation of the number of CFU use where an antibiotic has been used during production);
proceed as described for the pour-plate method. - Mycoplasma gallisepticum (where avian material has been
used during production or where the vaccine is intended
5-2-3 Most-probable-number method for use in poultry);
Prepare and dilute the sample using a method that has been - Mycoplasma hyorhinis (non-avian veterinary vaccines);
shown to be suitable as described in section 4. Incubate aH
- Mycoplasma orale (vaccines for human and veterinary use);
tubes at 30-35 cC for 3-5 days. Subculture if necessary, using
the procedure shown to be suitable. Record for each level of - Mycoplasma pneumoniae (vaccines for human use) or other
dilution the number of tubes showing microbial growth. suitable species of d-glucose fermenter such as
Determine the most probable number of micro-organisms Mycoplasma fermentans;
per gram or millilitre of the product to be examined from - Mycoplasma synoviae (where avian material has been used
Table 2.6.12.-3. during production or where the vaccine is intended for
use in poultry).
5-3 Interpretation 01 the results
The test strains are field iso lates having undergone a limited
The total aerobic microbial count (TAMC) is considered to
number of subcultures (not more than 15), and are stored
be equal to the number of CFU found using casein soya
frozen or freeze-dried. After cIoning, the strains are identified
bean digest agar; if colonies of fungi are detected on this
as being of the required species by comparison with type
medium, they are counted as part of the T AMC. The total
cultures, for example:
combined yeasts/mould count (TYMC) is considered to be
equal to the number of CFU found using Sabouraud- A. laidlawii NCTC 10116 CIP 75.27 ATCC 23206
dextrose agar; if colonies of bacteria are detected on this M. gallisepticum NCTC 10115 CIP 104967 ATCC 19610
medium, they are counted as part of the TYMe. When the M. f ermentans NCTCI0117 CIP 105680 ATCC 19989
TYMC is expected to exceed the acceptance criterion due to M. hyorhinis NCTC 10130 CIP 104968 ATCC 17981
the bacterial growth, Sabouraud-dextrose agar containing M. orale NCTC 10112 CIP 104969 ATCC 23714
antibiotics may be used. If the count is carried out by the M . pneumoniae NCTC 10119 CIP 103766 ATCC 15531
MPN method the calculated value is the TAMe. M. synoviae NCTC 10124 CIP 104970 ATCC 25204
When an acceptance criterion for microbiological quality is Acholeplasma laidlawii BRPJ Mycoplasma fennentans BRPJ
prescribed it is interpreted as follows: Mycoplasma hyorhinis BRPJ Mycoplasma orale BRP and
- 10 1 CFU: maxÍmum acceptable count = 20; Mycoplasma synoviae BRP are suitable for use as low-passage
- 102 CFU: maximum acceptable count = 200; reference strains.
- 103 CFU: maximum acceptable count = 2000, and so INCUBATION CONDITIONS
forth . Incubate liquid media in tightly stoppered containers at
The recommended solutions and media are described in 35-38 ce. Incubate solid media in microaerophilic conditions
general chapter 2.6.13. (nitrogen containing 5-10 per cent of carbon dioxide and
sufficient humidity to prevent desiccation of the agar surface)
at 35-38 0e.
2014 Appendix XVI B V -A4 71
NUTRITIVE PROPERTIES rotation. The test micro-organisms used are those listed
Carry out the test jor nut1itive propenies jor each new batch oj under Choice of culture media.
medium. Inoculate the chosen media with the appropriate test INTERPRETATION OF RESULTS
micro-organisms; use not more than 100 CFU (colony- At the end of the prescribed incubation period, examine all
forming units) per 60 mm diameter plate containing 9 mL of inoculated solid media microscopically for the presence of
solid medium and per 100 mL container of liquid medium; mycoplasma colonies. The product complies with the test if
use a separate plate and 'container for each species of micro- growth of typical mycoplasma colonies has not occurred.
organismo Incubate the media and make subcultures from The product does not comply with the test if growth of
0.2 mL of liquid medium to solid medium at the specified typical mycoplasma colonies has occurred on any of the solid
intervals (se e below under Test for mycoplasmas in the media. The test is invalid if 1 or more of the positive controls
product to be examined). The solid medium complies with do not show growth of mycoplasmas on at least 1 subculture
the test if adequate growth is found for each test microplate. The test is invalid if 1 or more of the negative controls
organism (growth obtained does not differ by a factor greater show growth of mycoplasmas. If suspect colonies are
than 5 from the value calculated with respect ro the observed, a suitable validated method may be used ro
inoculum). The liquid medium complies with the test if determine whether they are due ro mycoplasmas.
growth on agar plates subcultured from the broth is found
The jollowing section is published jor injormation.
for at least 1 sub culture for each test micro-organismo
INHIBITORY SUBSTANCES Recommended media for the culture method
The test for inhibitory substances is carried out once for a The following media are recommended. Other media may be
given product and is repeated whenever there is a change in used, provided that their ability ro sustain the growth of
production method that may affect the detection of mycoplasmas has been demonstrated on each batch in the
mycoplasmas. presence and absence of the product ro be examined.
To demonstrate absence of inhibitory substances, carry out HAYFLICK MEDIA (RECOMMENDED FOR THE GENERAL
the test for nutritive properties in the presence and absence DETECTION OF MYCOPLASMAS)
of the product to be examined. If growth of a test micro- Liquid medium
organism occurs more than 1 subculture sooner in the Beef heart infusion broth (1) 90.0 mL
absence of the product ro be examined than in its presence, Horse serum (unheated) 20.0 mL
or if pi ates directly inoculated with the product ro be Yeast extract (250 gIL) 10.0 mL
examined have fewer than 1/5 of the number of colonies of Phenol red (0.6 giL solution) 5.0 mL
those inoculated without the product ro be examined, Penicillin (20 000 IU/mL) 0.25 mL
inhibirory substances are present and they must be neutralised Deoxyribonueleic acid (2 gIL solution) 1.2 mL
or their effect otherwise countered, for example by passage in Adjust to pH 7.8.
substrates not containing inhibirors or dilution in a larger
volume of medium before the test. If dilution is used, larger Solid medium
rriedium volumes may be used or the inoculum volume may
Prepare as described aboye replacing beef heart infusion
be divided among several 100 mL ftasks . The effectiveness of
broth by beef heart infusion agar containing 15 gIL of agar.
the neutralisation or other process is checked by repeating the
test for inhibitory substances after neutralisation. FREY MEDIA (RECOMMENDED FOR THE DETECTION OF M.
SYNOVIAE)
TEST FOR MYCOPLASMAS IN THE PRODUCT TO BE
EXAMINED Liquid medium
Inoculate 10 mL of the product to be examined per 100 mL Beef heart infusion broth (1) 90.0 mL
of each liquid medium. If it has been found that a significant Essential vitamins (2) 0.025 mL
pH change occurs upon the addition of the product to be Glucose monohydrate (500 gIL solution) 2.0mL
examined, the liquid medium is resrored ro its original pH Swine serum (inactivated at 56 oC for 30 min) 12.0 mL
value by the addition of a solution of either sodium ~-Nicotinamide adenine dinueleotide (10 gIL 1.0 mL
hydroxide or hydrochloric acid. Inoculate 0.2 mL of the solution)
product to be examined on each plate of each solid medium. Cysteine hydrochloride (10 gIL solution) 1.0 mL
Incubate liquid media for 20-21 days. Incubate solid media Phenol red (0.6 gIL solution) 5.0 mL
for not les s than 14 days, except those corresponding ro the Penicillin (20 000 IU/mL) 0.25 mL
20-21 day sub culture, which are incubated for 7 days. At the
Mix the solutions of ~-nicotinamide adenine dinueleotide and
same time incubate an uninoculated 100 mL portion of each
cysteine hydrochloride and after 10 min add to the other
liquid medium and agar plates, as a negative control.
ingredients. Adjust ro pH 7.8.
On days 2-4 after inoculation, subculture each liquid
medium by inoculating 0.2 mL on at least 1 plate of each Solid medium
solid medium. Repeat the procedure between the 6th and
Beef heart infusion broth (1) 90.0 mL
8th days, again between the 13th and 15th days and again
Agar, purified (3) 1.4 g
between the 19th and 21 st days of the test. Observe the
liquid media every 2 or 3 days and if a colour change occurs, Adjust to pH 7.8, sterilise by autoelaving then add:
sub culture. If a liquid medium shows bacterial or fungal Essential vitamins (2) 0.025 mL
contamination, the test is invalido The test is valid if at least Glucose monohydrate (500 gIL solution) 2.0 mL
1 plate per medium and per inoculation day can be read. Swine serum (unheated) 12.0 mL
Inelude in the test positive controls prepared by inoculation ~-Nicotinamide adenine dinueleotide (10 gIL 1.0 mL
of not more than 100 CFU of at least 1 test micro-organism solution)
on agar medium or into broth medium . Where the test for Cysteine hydrochloride (10 gIL solution) 1.0 mL
mycoplasmas is carried out regularly and where possible, it is Phenol red (0.6 giL solution) 5.0 mL
recommended to use the test micro-organisms in regular Penicillin (20 000 IU/mL) 0.25 mL
FRlIS MEDIA (RECOMMENDED FOR THE DETECTION OF (5) Brain heart infusion
NON-AVIAN MYCOPLASMAS) Calf-brain infusion 200 g
Beef-heart infusion 250 g
Liquid medium Proteose peptone 10 g
Hanks' balanced salt solution (modified) (4) 800 mL Glucose monohydrate 2g
Distilled water 67mL Sodium chloride 5g
Brain heart infusion (5) 135 mL Disodium hydrogen phosphate, anhydrous 2.5 g
PPLO Broth (6) 248 mL Distilled water to 1000 mL
Yeast extract (170 gIL) 60mL (6) PPLO broth
Bacitracin 250 mg
Beef-heart infusion 50 g
Meticillin 250 mg
Peptone 10 g
Phenol red (5 giL) 4.5 mL
Sodium chloride 5g
Horse serum 165 mL
Distilled water to 1000 mL
Swine serum 165 mL
Adjust to pH 7.40-7.45. Indicator cell culture method
Solid medium Cell cultures are stained with a fluorescent dye that binds to
DNA. Mycoplasmas are detected by their characteristic
Hanks' balanced salt solution (modified) (4) 200 mL
particulate or filamentous pattern of fluorescence on the cell
DEAE-dextran 200 mg
surface and, if contamination is heavy, in surrounding areas.
Agar, purified (3) 15.65 g
Mitochondria in the cytoplasm may be stained but are readily
Mix well and sterilise by autoc1aving. Cool to 100 oc. Add to distinguished from mycoplasmas.
1740 mL of liquid medium as described aboye. If for viral suspensions the interpretation of results is affected
(1) Beef hean infusion broth by marked cytopathic effects, the virus may be neutralised
Beef heart (for preparation of the infusion) 500 g using a specific antiserum that has no inhibitory effects on
Peptone 10 g mycoplasmas or a cell culture substrate that does not allow
Sodium chloride 5g growth of the virus may be used. To demonstrate the
Distilled water to 1000 mL absence of inhibitory effects of serum, carry out the positive
control tests in the presence and absence of the antiserum.
Sterilise by autoc1aving.
VERIFICATION OF THE SUBSTRATE
(2) Essential vitamins
Use Vero cells or another cell culture (for example, the
Biotin 100 mg
production cellline) that is equivalent in effectiveness for
Calcium pantothenate 100 mg
detecting mycoplasmas. Test the effectiveness of the cells to
Choline chloride 100 mg
be used by applying the procedure shown below and
Folic acid 100 mg
inoculating not more than 100 CFU or CFU-like micro-
i-Inositol 200 mg
organisms of suitable reference strains of M. hyorhinis and
Nicotinamide 100 mg
M. orate. The following strains have been found to be
Pyridoxal hydrochloride 100 mg
suitable:
Riboflavine 10 mg
Thiamine hydrochloride 100 mg M. hyorhinis ATCC 29052
Distilled water to 1000 mL M.orale NCTC 10112 CIP 104969 ATCC 23714
(3) Agar, purified The cells are suitable if both reference strains are detected.
A highly refined agar for use in microbiology and The indicator cells must be subcultured without an antibiotic
immunology, prepared by an ion-exchange procedure that before use in the test.
results in a product having superior purity, c1arity and gel TEST METHOD
strength. It contains about: l. Seed the indicator cell culture at a suitable density (for
Water 12.2 per cent example,2 x 104 to 2 x 105 cells/mL, 4 x 10 3 to 2.5
Ash 1.5 per cent x 104 cells/cm 2) that will yield confluence after 3 days
Acid-insoluble ash 0.2 per cent of growth. Inoculate 1 mL of the product to be
Chlorine O examined into the cell culture vessel and incubate at
Phosphate (calculated as P 2 0 S) 0.3 per cent 35-38 oC .
Total nitro gen 0.3 per cent 2. After at least 3 days of incubation, when the cells have
Copper 8 ppm grown to confluence, make a sub culture on cover slips in
Iron 170 ppm suitable containers or on sorne other surface (for
Calcium 0.28 per cent example, chambered slides) suitable for the test
Magnesium 0.32 per cent procedure. Seed the cells at low density so that they
(4) Hanks' balanced salt solution (modified)
reach 50 per cent confluence after 3-5 days of
incubation. Complete confluence impairs visualisation of
Sodium chloride 6.4 g mycoplasmas after staining and must be avoided.
Potassium chloride 0.32 g
Magnesium sulfate heptahydrate 3. Remove the medium and rinse the indicator cells with
0.08 g
phosphate buffered saline pH 7.4 R, then add a suitable
Magnesium chloride hexahydrate 0.08 g
fixing solution (a freshly prepared mixture of 1 volume
Calcium chloride, anhydrous 0.112 g
Disodium hydrogen phosphate dihydrate of glacial acetic acid R and 3 volumes of methanol R is
0.0596 g
suitable when bisbenzimide R is used for staining).
Potassium dihydrogen phosphate, anhydrous 0.048 g
Distilled water to 800 mL
4. Remove the fixing solution and wash the cells with - A. laidlawii;
sterile water R. Dry the slides comp1etely if they are to - M. fermentans;
be stained more than 1 h later (particular care is needed - M. hyorhinis (where cell-culture enrichment is used, a
for staining of slides after drying owing to artefacts that fastidious strain such as ATCC 29052 is included);
may be produced).
- M. orale;
5. Add a suitable DNA stain and anow to stand for a
suitable time (bisbenzimide working solution R and a - M. pneumoniae or M. gallisepticum;
standing time of 10 min are suitable). - M. synoviae (where there is use of or exposure to avian
6. Remove the stain and rinse the monolayer with water R. material during production);
7. Mount each coverslip, where applicable (a mixture of - Mycoplasma arginini;
equal volumes of g1ycerol R and phosphate-citrate buffer - Spiroplasma cien· (where there is use of or exposure to
solution pH 5.5 R is suitable for mounting). Examine by insect or plant material during production).
fluorescence (for bisbenzimide stain a 330 nm/380 nm Demonstration of specificity requires the use of a suitable
excitation filter and an LP 440 nm barrier filter are range of bacterial species other than mycoplasmas. Bacterial
suitable) at 400 x magnification or greater. genera with close phylogenetic relation to mycoplasmas are
8. Compare the microscopic appearance of the test cultures most appropriate for this validation; these include
with that of the negative and positive controls, Clostridiurn, Lactobacillus and Streptococcus.
examining for extranuclear fluorescence. Mycoplasmas Comparability studies for use of NAT as an alternative
produce pinpoints or filaments over the indicator cell method For each mycoplasma test species:
cytoplasm. They may also produce pinpoints and - as an altemative to the culture method: the NAT test
filaments in the intercellular spaces. Multiple system must be shown to detect 10 CFU/mL;
microscopic fields are examined according to the - as an altemative to the indicator cell culture method: the
protocol established during validation. NAT test system must be shown to detect 100 CFU/mL;
INTERPRETATION OF RESULTS or an equivalent limit of detection in terms of the number of
The product to be examined complies with the test if copies of mycoplasma nucleic acid in the test sample (using
fluorescence typical of mycoplasmas is not presento The test suitable reference standards of mycoplasma nucleic acid) .
is invalid if the positive controls do not show fluorescence
CONTROLS
typical of mycoplasmas. The test is invalid if the negative
controls show fluorescence typical of mycoplasmas. Internal controls Intemal controls are necessary for
routine verification of absence of inhibition. The internal
Nucleic acid amplification techniques (NA T) control may contain the primer binding-site, or sorne other
suitable sequence may be used. It is preferably added to the
NAT (2.6.21) may be used for detection of mycoplasmas by
test material before isolating the nucleic acid and therefore
amplification of nucleic acids extracted from a test sample
acts as an overall control (extraction, reverse transcription,
with specific primers that reveal the presence of the target
amplification, detection) .
nucleic acid. NAT indicate the presence of a particular
nucleic acid sequence and not necessarily the presence of External controls The extemal positive control contains a
viable mycoplasmas. A number of different techniques are defined number of target-sequence copies or CFUs from 1
available. This general chapter does not prescribe a particular or more suitable species of mycoplasma chosen from those
method for the test. The procedure applied must be used during validation of the test conditions. 1 of the
validated as described, taking account of the guidelines positive controls is set close to the positive cut-off point to
presented at the end of this section. Where a commercial kit demonstrate that the expected sensitivity is achieved.
is used, certain elements of the validation may be carried out The external negative control contains no target sequence
by the manufacturer and information provided to the user but does not necessarily represent the same matrix as the test
but it must be remembered that full information on the article.
primers may not be available and that production of the kit INTERPRETATION OF RESULTS
may be modified or discontinued. The primers used may also amplify non-mycoplasmal
NAT are applied where prescribed in a monograph. They bacterial nucleic acid, leading to false positive results.
may also be used instead of the culture method and the Procedures are established at the time of validation for
indicator cen culture method after suitable validation. dealing with confirmation of positive results, where necessary.
Direct NAT can be applied in the presence of cytotoxic The following section is published for information.
material and where a rapid method is needed.
Validation of nucleic acid amplification techniques
Cell-culture enrichmentfollowed by NAT The test
(NAT) for the detection ofmycoplasmas: guidelines
sample and a suitable cell substrate (as described under the
indicator cen-culture method) are cultured together for a 1 SCOPE
suitable period; the nucleic acids are then extracted from Nucleic acid amplification techniques (NAT) are either
cells and supernatant and used for detection by NAT. qualitative or quantitative tests for the presence of nucleic
acid. For the detection of mycoplasma contamination of
VALIDATION
various samples such as vaccines and cell substrates,
Reference standards are required at various stages during qualitative tests are adequate and may be considered to be
validation and for use as controls during routine application limit tests.
of the test. The reference standards may be mycoplasmas or
nucleic acids. These guidelines describe methods to validate qualitative
nucleic acid amplification analytical procedures for assessing
For validation of the limit of detection, the following species mycoplasma contamination. They may also be applicable for
represent an optimal selection in terms of the frequency of real-time NAT used as limit tests for the control of
occurrence as contaminants and phylogenetic relationships: contaminants.
The 2 characteristics regarded as the most important for For establishment of the detection limit, a positive cut-off
validation of the analytical procedure are the specificity and point should be determined for the nucleic acid amplification
the detection limito In addition, the robustness of the analytical procedure. The positive cut-off point (as defined in
analytical procedure should be evaluated. general chapter 2.6.21) is the minimum number of target
For the purpose of this document, an analytical procedure is sequence copies per volume of sample that can be detected
defined as the complete procedure from extraction of nucleic in 95 per cent of test runs. This positive cut-off point is
acid to detection of the amplified products. influenced by the distribution of mycoplasmal genomes in the
individual samples being tested and by factors such as
Where commercial kits are used for part or all of the
enzyme efficiency, and can result in different 95 per cent cut-
analytical procedure, documented validation points already
off values for individual analytical test runS.
covered by the kit manufacturer can replace validation by the
user. Nevertheless, the performance of the kit with respect to To determine the positive cut-off point, a dilution series of
its intended use has to be demonstrated by the user characterised and calibrated (either in CFUs or nucleic acid
(e.g. detection limit, robustness, cross-detection of other copies) in-house working strains or EDQM standards should
classes of bacteria). be tested on different days to examine variation between test
runs .
NAT may be used as:
For validation of the limit of detection, the following species
- a complementary test (for example, for cytotoxic viral
represent an optimal selection in terms of the frequency of
suspensions) or for in-process control purposes;
occurrence as contaminants and phylogenetic relationships:
- an alternative method to replace an official method
- A. laidlawii;
(indicator cell culture method or culture method).
- M. fermentans;
These guidelines will thus separate these 2 objectives by
presenting first a guideline for the validation of the NAT - M. hyorhinis;
themselves, and second, a guideline for a comparability study - M. orale;
between NAT and official methods . '- M. pneumoniae or M. gallisepticum;
2 GUIDELINE FOR MYCOPLASMA NAT VALIDATION - M. synoviae (where there is use of or exposure to avian
3 parameters should be evaluated: specificity, detection limit material during production);
and robustness. - M. arginini;
2-1 Specificity Specificity is the ability to unequivocally - S. citri (where there is use of or exposure to insect or
assess target nucleic acid in the presence of components that plant material during production) .
may be expected to be presento
For each strain, at least 3 independent 10-fold dilution series
The specificity of NAT is dependent on the choice of should be tested, with a sufficient number of replicates at
primers, the choice of probe (for analysis of the final each dilution to give a total number of 24 test results for
product) and the stringency of the test conditions (for both each dilution, to enable a statistical analysis of the results.
the amplification and detection steps).
For example, a laboratory may test 3 dilution series on
The ability of the NAT to detect a large panel of different days with 8 replicates for each dilution, 4 dilution
mycoplasma species will depend on the choice of primers, series on different days with 6 replica tes for each dilution, or
pro bes and method parameters. This ability should be 6 dilution series on different days with 4 replicates for each
demonstrated using characterised reference panels dilution. In order to keep the number of dilutions at a
(e.g. reference strains provided by the EDQM). Since NAT manageable level, a preliminary test should be performed to
systems are usually based on a mix of primers, the theoretical obtain a preliminary value for the positive cut-off point
analysis of primers and pro bes by comparison with databas es (i.e. the highest dilution giving a positive signa!) . The range
is not recommended, beca use interpretation of the results of dilutions can then be chosen around the predetermined
may be quite complex and may not reflect the experimental preliminary cut-off point. The concentration of mycoplasmas
results. (CFUs or copies) that can be detected in 95 per cent of test
Moreover, as it is likely that the primers will detect other runs can then be calculated using an appropriate statistical
bacterial species, the potential cross-detection should be evaluation.
documented in the validation study. Bacterial genera such as These results may also serve to evaluate the variability of the
gram-positive bacteria with close phylogenetic relation to analytical procedure.
mycoplasmas are most appropriate for this validation; these
2-3 Robustness The robustness of an analytical procedure
include Clostridium, Lacwbacillus and Strepwcoccus. However,
is a measure of its capacity to remain unaffected by small
this is not an exhaustive list and species to be tested will
but deliberate variations in method parameters, and provides
depend on the theoretical ability (based on primers/probes
an indication of its reliability during normal usage.
sequences) of the NAT system to detect such other species.
The evaluation of robustness should be considered during
Based on the results from this validation of the specificity, if
the development phase. It should show the reliability of the
a gap in the specificity of the method is identified (such as
analytical procedure with respect to deliberate variations in
detection of non-mycoplasmal bacterial nucleic acid), an
method parameters. For NAT, small variations in the
appropriate strategy must be proposed in the validation study
method parameters can be crucial. However, the robustness
to allow interpretation of positive results on a routine basis.
of the method can be demonstrated during its development
For example, a second test may be performed using an
when small variations in the concentrations of reagents
alternative method without this specificity gap or using an
(e.g. MgCl z, primers or deoxyribonucleotides) are tested.
official method.
Modifications of extraction kits or extraction procedures as
2-2 Detection limito The detection limit of an individual well as different thermal cycler types may also be evaluated.
analytical procedure is the lowest amount of target nucleic
Finally, robustness of the method can be evaluated through
acid in a sample that can be detected but not necessarily
collaborative studies.
quantitated as an exact value.
3 GUIDELINE FOR COMPARABILITY STUDY 5. Extraneous Agents in Viral Vaccines

NAT may be used instead of official methods (indicator cell (Ph. Eur. methad 2.6.16)
culture method ami/or culture method) . In this case a In those tests that require prior neutralisation of the virus,
comparability study should be carried out. This comparability use specific antibodies of non-human, non-simian originó
study should include mainly a comparison of the respective if the virus has been propagated in avian tissues, the
detection limits of the altemative method and official antibodies must also be of non-avian origino To prepare
methods. However, specificity (mycoplasma panel detected, antiserum, use an immunising antigen produced in ceH
putative false positive results) should also be considered. culture from a species different from that used for the
For the detection limit, acceptability criteria are defined as production of the vaccine and free from extraneous agents.
follows : Where the use of SPF eggs is prescribed, the eggs are
- if the altemative method is proposed 10 replace the culture obtained from a flock free from specified pathogens (5.2.2) .
method, the NAT system must be shown to detect 10 Virus seed lot
CFU/mL for each mycoplasma test species described in Take samples of the virus seed lot at the time of harvesting
paragraph 2-2; and, if they are not tested immediately, keep them at a
- if the altemative method is proposed to replace the temperature below - 40 oc.
indicator cell culture method, the NAT system must be Adult mice Inoculate each of not fewer than 10 adult
shown to detect 100 CFU/mL for each mycoplasma test mice, each weighing 15-20 g, intracerebrally with 0.03 mL
species described in paragraph 2-2. and intraperitoneally with 0.5 mL of the virus seed 101.
For both cases, suitable standards calibrated for the number Observe the mice for at least 21 days. Carry out an autopsy
of nucleic acid copies and the number of CFUs may be used of all mice that die after the first 24 h of the test or that
for establishing that these acceptability criteria are reached. show signs of illness, and examine for evidence of viral
The relation between CFUs and nucleic acid copies for the infection, both by direct macros copie al observation and by
reference preparations should be previously established 10 subinoculation of appropriate tissue suspensions by the
compare the performance of the altemative NAT method intracerebral and intraperitoneal routes into not fewer than 5
with the performance of the official methods. additional mice, which are observed for 21 days. The virus
1 of the following 2 strategies can be used to perform this seed lot complies with the test if no mouse shows evidence
comparability study: of infection attributable to the seed lot. The test is not valid
- perform the NAT altemative method in parallel with the unless at least 80 per cent of the original inoculated mice
official method(s) to evaluate simultaneously the detection survive the observation periodo
limit of both methods using the same samples of Suckling mice Inoculate each of not fewer than 20 mice,
calibra ted strains; each less than 24 h old, intracerebrally with 0.01 mL and
- compare the performance of the NAT altemative method intraperitoneally with at least 0.1 mL of the virus seed lot.
using previously obtained data from official method Observe the mice daily for at least 14 days. Carry out an
validation. In this case, calibration of standards used for autopsy of aH mice that die after the first 24 h of the test or
both validations as well as their stabilities should be that show signs of illness, and examine for evidence of viral
documented carefully. infection, both by direct macroscopical observation and by
subinoculation of appropriate tissue suspensions by the
Comparability study reports should describe all the validation
intracerebral and intraperitoneal routes into not fewer than 5
elements described in section 2 (specificity, limit of detection
additional suckling mice, which are observed daily for
and variability, as well as robusmess) in order 10 assess all
14 days. The virus seed lot passes the test if no mouse shows
the advantages and/or disadvantages of the altemative NAT
evidence of infection attributable to the seed lot. The test is
method compared to official methods.
not valid unless at least 80 per cent of the original inoculated
4. Mycobacteria mice survive the observation periodo
Guinea-pigs Inoculate intraperitoneally into each of not
fewer than 5 guinea pigs, each weighing 350-450 g, 5.0 mL
If the sample to be examined may be contaminated by
of the virus seed lot. Observe the animals for at least 42 days
micro-organisms other than mycobacteria, treat it with a
for signs of disease. Carry out an autopsy of aH guinea-pigs
suitable decontamination solution, such as acety1cysteine-
that die after the first 24 h of the test or that show signs of
sodium hydroxide solution or sodium laurilsulfate solution.
illness, and examine macroscopically; examine the tissues
Inoculate 0.2 mL of the sample in triplica te onto each of 2 both microscopically and culturally for evidence of infection.
suitable solid media (L6wenstein-Jensen medium and Euthanise animals that survive the observation period and
Middlebrook 7HI0 medium are considered suitable). examine in a similar manner. The virus seed lot passes the
Inoculate 0.5 mL in triplicate into a suitable liquid medium. test if no guinea-pig shows evidence of infection attributable
Incubate aH media at 37 oC for 56 days. to the seed 101. The test is not valid unless at least
Establish the fertility of the media in the presence of the 80 per cent of the guinea-pigs survive the observation periodo
preparation 10 be examined by inoculation of a suitable strain Spiroplasmas Virus seed lots produced in insect cells are
of a Mycabacterium sp. su eh as BCG and if necessary use a demonstrated by a validated method approved by the
suitable neutralising substance. competent authority to be free of spiroplasmas.
If contaminating micro-organisms develop during the first
Virus seed lot and virus harvests
8 days of incubation, repeat the test and carry out at the
same time a bacteriological sterility test. Take samples at the time of harvesting and, if not tested
immediately, keep them at a temperature below - 40 oc.
If at the end of the incubation time no growth of
mycobacteria occurs in any of the test media, the preparation Bacterial and fungal sterility A 10 mL sample complies
complies with the test. with the test for sterility (2.6.1).
V-A476 Appendix XVI e 2014
Mycoplasmas A 10 mL sample complies with the test for been stored at 5 ± 3 oC for not more than 7 days. Read
mycoplasmas (2.6.7). half of the cultures after incubation at 5 ± 3 cC for 30 min
Mycobacteria (2. 6.2) A 5 mL sample is tested for the and the other half after incubation at 20-25 oC for 30 mino
presence of Mycobacterium spp. by culture methods known to No evidence of haemadsorbing agents is found.
be sensitive for the detection of these organisms. Tests in cell cultures for other extraneous agents Pool
Test in cell culture for other extraneous agents the supematant fluids from the control cells and examine for
Neutralised samples equivalent, unless otherwise prescribed, the presence of extraneous agents by inoculation of simian
to 500 human doses of vaccine or 50 mL, whichever is the kidney and human cell cultures. If the virus is grown in a
greater, are tested for the presence of extraneous agents by marnmalian cell system other than simian or human, cells of
inoculation into continuous simian kidney and human cell that species, but from a separate batch, are also inoculated.
cultures. If the virus is grown in simian or human cells, the In each cell system, at least 5 mL is tested. Incubate the
neutralised virus harvest is tested on a separate culture of inoculated cultures at 36 ± 1 oC and observe for a period of
these cells. If the virus is grown in a mammalian or avian 14 days . No evidence of extraneous agents is found.
cell system other than simian or human, cells of that species, If the production cell culture is maintained at a temperature
but from a separate batch, are also inoculated. The cells are different from 36 ± 1 °C, a supplementary test for
incubated at 36 ± 1 oC and observed for a period of extraneous agents is carried out at the production
14 days. The virus seed lot or harvest passes the tests if none temperature using the same type of cells as used for growth
of the cell cultures show evidence of the presence of any of the virus.
extraneous agents. The test is not valid unless at least If the virus is grown in insect cells the pooled supematant is
80 per cent of the cell cultures remain viable. also inoculated into at least one cell culture different from
A vian viruses (onIy required for virus seed lot that used in production and perrnissible to insect viruses, and
propagated in avian tissues and for virus harvest that allows detection of human arboviruses. The cells are
propagated in primary avian tissues) Neutralise a incubated at 27 ± 1 oC for 14 days. No evidence of
sample equivalent to 100 human doses of vaccine or 10 mL, extraneous agents is found .
whichever is the greater. Using 0.5 mL per egg, inoculate a Aviao leucosis viruses (required only ifthe virus is
group of fertilised SPF eggs, 9-11 days old, by the allantoic propagated in primary avian tissues) Carry out a test
route and a second group, 5-7 days old, into the yolk saco for avian leucosis viruses using 5 mL of the supematant fluid
Incubate for 7 days. The virus seed lot or harvest complies from the control cells.
with the test if the allantoic and yolk sac fluids show no sign
of the presence of any haemagglutinating agent and if all Control eggs
embryos and chorio-allantoic membranes, examined for gross Haemagglutinating agents Examine 0.25 mL of the
pathology, are nortnal. The test is not valid unless at least allantoic fluid from each egg for haemagglutinating agents by
80 per cent of the inoculated eggs survive for 7 days. mixing directly with chicken red blood cells and after a
Insect viruses (only required for virus propagated in passage in SPF eggs carried out as follows: inoculate a 5 mL
iosect cells) Neutralised samples equivalent, unless sample of the pooled amniotic fluids from the control eggs in
otherwise prescribed, to 500 human doses of vaccine or 0.5 mL volumes into the allantoic cavity and into the
amniotic cavity of SPF eggs. The control eggs comply with
50 mL, whichever is the greater, are tested for the presence
the test if no evidence of the presence of haemagglutinating
of extraneous agents by inoculation into at least one cell
agents is found in either test.
culture different from that used in production and
permissible to insect viruses, and that allows detection of Avian leucosis viruses Use a 10 mL sample of the
human arboviruses. The choice of cells is approved by the pooled amniotic fluids from the control eggs. Carry out
competent authority and takes into account the origin of the amplification by 5 passages in leucosis-free chick-embryo cell
production cells and the likely contaminants that may be cultures; carry out a test for avian leucosis using cells from .
detected by the chosen cells. The cells are incubated at the 5th passage. The control eggs comply with the test if no
27 ± 1 oC and observed for a period of 14 days. The virus evidence of the presence of avian leucosis viruses is found.
seed lot or harvest passes the tests if none of the cell cultures Other extraneous agents Inoculate 5 mL samples of the
show evidence of the presence of any extraneous agents. pooled amniotic fluids from the control eggs into human and
The test is not valid unless at least 80 per cent of the cell simian cell cultures. Observe the cell cultures for 14 days.
cultures remain viable. The control eggs comply with the test if no evidence of the
Production cell culture: control cells presence of extraneous agents is found. The test is not valid
unless 80 per cent of the inoculated cultures survive to the
Examine the control cells microscopically for freedom from end of the. observation periodo
any virus causing cytopathic degeneration throughout the
time of incubation of the inoculated production cell cultures
or for not less than 14 days beyond the time of inoculation of
the production vessels, whichever is the longer. The test is C. Efficacy of Antimicrobial Preservation
not valid unless at least 80 per cent of the control cell
(Ph. Eur. general text 5. 1.3)
cultures survive to the end of the observation periodo
If a pharmaceutical preparation do es not itself have adequate
At 14 days or at the time of the last virus harvest, whichever antimicrobial activity, antimicrobial preservatives may be
is the longer, carry out the tests described below. added, particularly to aqueous preparations, to prevent
Haemadsorbing viruses Examine not fewer than proliferation or to limit microbial contamination which,
25 per cent of the control cultures for the presence of during normal conditions of storage and use, particularly for
haemadsorbing viruses by the addition of guinea-pig red multidose containers, could occur in a product and present a
blood cells. If the test for haemadsorbing virus es is not hazard to the patient from infection and spoilage of the
feasible, carry out a test for haemagglutination viruses. If the preparation. Antimicrobial preservatives must not be used as
guinea-pig red blood cells have been stored, they shall have a substitute for good manufacturing practice.
2014 Appendix XVI e v -A4 77
The efficacy of an antimicrobial preservative may be To harvest the bacterial and C. albicans cultures, use a sterile
enhanced or diminished by the active constituent of the suspending fluid, containing 9 gIL of sodium chloride R, for
preparation or by the formulation in which it is incorporated dispersal and transfer of the surface growth into a suitable
or by the container and closure used. The antimicrobial vessel. Add sufficient suspending fluid to reduce the
activity of the preparation in its final container is investigated microbial count to about 108 micro-organisms per millilitre.
over the period of validity to ensure that such activity has not To harvest the A. brasiliensis culture, use a sterile suspending
been impaired by storage. Such investigations may be carried fluid containing 9 gIL of sodium chlonde R and 0.5 gIL of
out on samples removed from the final container immediately polysorbate 80 R and adjust the spore count to about 108 per
prior to testing. millilitre by adding the same solution.
During development of a pharmaceutical preparation, it shall Remove immediately a suitable sample from each suspension
be demonstrated that the antimicrobial activity of the and determine the number of colony-forming units per
preparation as such or, if necessary, with the addition of a millilitre in each suspension by plate count or membrane
suitable preservative or preservatives provides adequate filtration (2.6. 12) . This value serves to determine the
protection from adverse effects that may arise from microbial inoculum and the baseline to use in the test. The suspensions
contamination or proliferation during storage and use of the shall be used immediately.
preparation.
The efficacy of the antimicrobial activity may be METHOD
demonstrated by the test described below. The test is not To count the viable micro-organisms in the inoculated
intended to be used for routine control purposes. products, use the agar medium used for the initial cultivation
of the respective micro-organisms.
Test for efficacy of antimicrobial preservation Inoculate a series of containers of the product to be
The test consists of challenging the preparation, wherever examined, each with a suspension of one of the test
possible in its final container, with a prescribed inoculum of organisms to give an inoculum of 10 5 to 10 6 micro-organisms
suitable micro-organisms, storing the inoculated preparation per millilitre or per gram of the preparation. The volume of
at a prescribed temperature, withdrawing samples from the the suspension of inoculum does not exceed 1 per cent of the
container at specified intervals of time and counting the volume of the product. Mix thoroughly to ensure
organisms in the samples so removed. homogeneous distribution.
The preservative properties of the preparation are adequate Maintain the inoculated product at 20-25 oC, protected from
if, in the conditions of the test, there is a significant fall or no light. Remove a suitable sample from each container,
increase, as appropriate, in the number of micro-organisms in typically 1 mL or 1 g, at zero hour and at appropriate
the inoculated preparation after the times and at the intervals according to the type of the product and determine
temperatures prescribed. The acceptance criteria, in terms of the number of viable micro-organisms by plate count or
decrease in the number of micro-organisms with time, vary membrane filtration (2.6.12) . Ensure that any residual
for different types of preparations according to the degree of antimicrobial activity of the product is eliminated by dilution,
protection intended (se e Tables 5.1.3 .-112/3) . by filtration or by the use of a specific inactivator. When
dilution procedures are used, due allowance is made for the
Test micro-organisms reduced sensitivity in the recovery of small numbers of viable
micro-organisms. When a specific inactivator is used, the
Pseudo monas aeruginosa ATCC 9027 ; NCIMB 8626; CIP 82.118.
ability of the system to support the growth of the test
Staphylococcus aureus ATCC 6538; NCTC 10788; organisms is confirmed by the use of appropriate controls.
NCIMB 9518; CIP 4.83.
The procedure is validated to verify its ability to demonstrate
Candida albicans ATCC 10231; NCPF 3179; IP 48.72.
the required reduction in count of viable micro-organisms.
Aspergillus brasiliensis ATCC 16404; IMI 149007; IP 1431.83.
Acceptance of Criteria
Single-strain challenges are used and the designated micro- The criteria for evaluation of antimicrobial activity are given
organisms are supplemented, where appropriate, by other in Tables 5.1.3.-112/3 in terms ofthe log reduction in the
strains or species that may represent likely contaminants to number of viable micro-organisms against the value obtained
the preparation. It is recommended, for example, that for the inoculum.
Eschen·chia coli (ATCC 8739; NCIMB 8545; CIP 53.126) is
used for all oral preparations and Zygosaccharomyces rouxii
Table 5.1.3.-1. - Parenteral preparations, eye preparations,
(NCYC 381; IP 2021.92) for oral preparations containing a intrauterine preparations and íntramammary preparations
high concentration of sugar.
Lag reductian
Preparation of inoculum
6h 24 h 7d 14 d 28d
Preparatory to the test, inoculate the surface of casein soya
Bacteria A 2 3 NR
bean digest agar (2.6.12) for bacteria or Sabouraud-dextrose
agar without the addition of antibiotics (2.6.12) for fungi, B 3 NI
with the recently grown stock culture of each of the specified Fungi A NI
micro-organisms. Incubate the bacterial cultures at 30-35 oC
B NI
for 18-24 h, the culture of C. albicans at 20-25 oC for 48 h,
and the culture of A. brasiliensis at 20-25 oC for 1 week or NR: no recovery.
until good sporulation is obtained. Subcultures may be NI: no increase in number of viable micro-organisms compared lo the
previous reading.
needed after revival before the micro-organism is in its
optimal sta te, but it is recommended that their number be
kept to a minimum. The A criteria express the recommended efficacy to be
achieved. In justified cases where the A criteria cannot be
V-A478 Appendix XVI D 2014
attained, for example for reasons of an inereased risk of

adverse reaetions, the B eriteria must be satisfied.
D. Microbiological Quality of Non-sterile
Pharmaceutical Preparations and
Table 5.1.3.-2. - Ear preparations, nasal preparations, Substances for Pharmaceutical Use 1
preparations for cutaneous application and preparations for
inhalation (Ph. Eur. general text 5.1.4)
The presenee of eertain miero-organisms in non-sterile
Log reduction
preparations may have the potential to reduce or even
2d 7d 14 d 28 d inaetivate the therapeutie activity of the produet and has a
Bacteria A 2 3 NI potential to adversely affect the health of the patient.
Manufacturers therefore have to ensure a low bioburden of
B 3 NI
finished dosage forms by implementing eurrent guidelines on
Fungi A 2 NI Good Manufaeturing Practiee during the manufacture,
B NI storage and distribution of pharrnaeeutieal preparations.
NI: no increase in number of viable micro-organisms compared to the
Microbial examination of non-sterile products is perforrned
previous reading. aeeording to the methods given in general ehapters 2.6. 12
and 2.6.13. Aeeeptanee eriteria for non-sterile pharrnaeeutieal
The A eriteria express the reeommended effieaey to be produets based upon the total aerobie microbial eount
aehieved. In justified cases where the A eriteria eannot be (TAMC) and the total eombined yeasts/moulds eount
attained, for example for reasons of an inereased risk of (TYMC) are given in Tables 5.1.4.-1 and 5.1.4.-2.
adverse reaetions, the B criteria must be satisfied. Aeeeptanee eriteria are based on individual results or on the
average of replicate eounts when replieate eounts are
perforrned (e.g. direct plating methods) .
Table 5.l.3.-3. - Oral preparations, oromucosal preparations When an aeeeptanee eriterion for microbiologieal quality is
and rectal preparations
preseribed it is interpreted as follows:
Log reduction
- 10 1 CFU: maximum aeeeptable eount = 20;
14 d 28 d - 10 2 CFU: maximum acceptable eount = 200;
Bacteria 3 NI - 103 CFU: maximum aeeeptable eount = 2000, and so
Fungi NI forth.
NI: no increase in number of viable micro-organisms compared to the Table 5.1.4.-1 includes a list ofspeeified miero-organisms for
previous reading. whieh aceeptanee eriteria are seto The list is not neeessarily
exhaustive and for a given preparation it may be neeessary to
The aboye eriteria express the reeommended efficaey to be test for other miero-organisms depending on the nature of
aehieved. the starting materials and the manufaeturing proeess.
Table 5.1.4.-1. - Acceptance criteria for microbiological quality of non-sterile dosage forms
TAMC TYMC
Route oC administration Specified micro-organisms
(CFU/g or CFU/mL) (CFU/gor CFU/mL)
Non-aqueous preparations for oral use lO' !O' Absence of Escherichia coli (1 g or 1 mL)
Aqueous preparations for oral use !O' !O' Absence of Escherichia coli O g or 1 mL)
Rectal use 103 la'

Oromucosal use
Gingival use
Absence of Staphylococcus aureus (1 g or 1 mL)
Cutaneous use !O' !O'
Absence of Pseudomonas aeruginosa O g or 1 mL)
Nasal use
Auricular use
Absence of Pseudomonas aeruginosa (1 g or 1 mL)
Vaginal use !O' la' Absence of Staphylococcus aureus (1 g or 1 mL)
Absence of Gandida albicans (1 g or 1 mL)
Transdermal patches (limits for one patch Absence of Staphylococcus aureus O patch)
lO' lO'
including adhesive layer and backing) Absence of Pseudomonas aeruginosa (1 patch)
Absence of Staphylococcus aureus O g or 1 mL)
Inhalation use (special requirements apply to Absence of Pseudomonas aeruginosa O g or 1 mL)
liquid preparations for nebulisation)
!O' lO'
Absence of bile·tolerant gram-negative bacteria
(1 g or 1 mL)
• Special Ph. Eur. provision for oral dosage forms Not more than lO' CFU of bile-tolerant gram-negative
containing raw materials of natural (animal, bacteria (1 g or 1 mL)
vegetal or mineral) origin for which antimicrobial Absence of Salmonella 00 g or 10 mL)
pretreatment is not feasible and for which the
10' !O'
competent authority accepts TAMC of the raw Absence of Escherichia coli (1 g or 1 mL)
material exceeding 103 CFU/ g or CFU/ mL. Absence of Staphylococcus aureus O g or 1 mL).
I This chapter has undergone phannacopoeial hannonisation. See chapter
5.8. Phannacopoeial harmonisation.

2014 Appendix XVI E V-A479
If it has been shown that none of the prescribed tests will Each batch of medium is tested by the supplier and/or the
allow val id enumeration of micro-organisms at the leve! user for its growth-promoting capacities by inoculating
prescribed, a validated method with a limit of detection as duplicate test containers of each medium with 10-100 viable
close as possible to the indicated acceptance criterion is used. micro-organisms of each of the strains listed in
In addition to the micro-organisms listed in Table 5.1.4.-1, Table 2.6.27.-1, and incubating for either 7 days for
the significance of other micro-organisms recovered is automated detection or 14 days for visual detection of
evaluated in terms of: microbial growth at 35-37 oC. The test media are satisfactory
- use of the product: hazard varies according to the route of if there is clear evidence of growth in all inoculated media
administration (eye, nose, respiratory tract); containers within this periodo
- nature of the product: its ability to support growth, the
presence of adequate antirnicrobial preservation; Table 2.6.27.-1. - Micro-organisms used for growth promotion
- method of application; Aerobic medium
- intended recipient: risk may differ for neonates, infants, Staphylococcus aureus for example, ATCC 6538, CIP 4.83,
the debilitated; NCTC 10788, NCIMB 9518
- use of immunosuppressive agents, corticosteroids;
Bacillus subtilis for example, ATCC 6633, CIP 52.62,
- presence of di seas e, wounds, organ damage. NCIMB 8054
Where warranted, a risk-based assessment of the relevant Pseudomonas aeruginosa for example, ATCC 9027, NCIMB 8626,
factors is conducted by perso=e! with specialised training in CIP 82.118
microbiology and the interpretation of microbiological data.
For raw materials, the assessment takes account of processing Candida albicans for example, ATCC 10231, IP 48.72, NCPF
3179
to which the product is subjected, the current technology of
Aspergillus brasiliensis for example, ATCC 16404, IP 1431.83, IMI
testing and the availability of materials of the desired quality. 149007
Anaerobic medium
Table 5.1.4.-2. - Acceptance criteria for microbiological quality
of non-sterile substances for pharmaceutical use Clostridium sporogenes for example, ATCC 19404, CIP 79.3, NCTC
532 or ATCC 11437
TAMC TYMC
( CFU/g or CFU/mL) ( CFU/g or CFU/mL) Bacteroides fragilis for example, ATCC 25285, CIP 77.16,
NCTC 9343
Substances for
pharmaceutical use
103 lO'
METHOD VALIDATION
Recommended acceptance criteria for microbiological quality of Depending on the type of product, its method of preparation,
herbal medicinal products for oral use are given in general chapter the inoculum volume used and the type of test system, the
5.1.8. need for validation in the presence of the type of preparation
to be examined must be considered. Unless otherwise
justified and authorised, the test system is validated with
respect to specificity (absence of false positive results),
sensitivity (limit of detection) and reproducibility. During
E. Microbiological Control of Cellular validation, particularly to determine the limit of detection,
Products the test is carried out using the preparation deliberately
(Ph. Eur. method 2.6.27) contaminated to different degrees with the following micro-
organisms, chosen for the likelihood of contamination and
This test has been shown to be preferable to the test for
their growth requirements:
sterility (2.6.1) for certain cellular products, since it has
better sensitivity, has a broader range, and is more rapid. It is - Aspergillus brasiliensis, for example, ATCC 16404, IP
applied instead of the test for sterility (2.6.1) where 1431.83,IM! 149007;
prescribed in a monograph. It may be carried out manually - Bacillus subtilis, for example, ATCC 6633, CIP 52.62,
or using an automated system. NCIMB 8054;
GENERAL PRECAUTIONS - Gandida albicans, for example, ATCC 10231 , IP 48 .72,
The test is carried out under aseptic conditions according to NCPF 3179;
current regulations for potentially infective material. - Glostridium sporogenes, for example, ATCC 19404, CIP
The precautions taken to avoid contamination are such that 79.3, NCTC 532 or ATCC 11437;
they do not affect any micro-organisms that are to be - Propionibacterium acnes, for example, ATCC 11827;
revealed in the test. The test is performed under working - Pseudomonas aeruginosa, for example, ATCC 9027,
conditions that are monitored regularly by appropriate NCIMB 8626, CIP 82.118;
sampling of the working area and by carrying out appropriate - Staphylococcus aureus, for example, ATCC 6538, CIP
controls. 4.83, NCTC 10788, NCIMB 9518;
GROWTH PROMOTION TEST - Streptococcus pyogenes, for example, ATCC 19615, CIP
Use at least 2 suitable enriched culture media (for example, 1042.26, NCIMB 13285;
blood culture media) intended for detection of fungi and
- Yersinia enterocolitica, for example, ATCC 9610, CIP
aerobic and anaerobic bacteria.
80.27, NCTC 12982.
Confirm the sterility of each batch of medium by the
It may be necessary to modify the list of micro-organisms
incubation of representative containers at 35-37 oC for not
depending on the origin of the cells and any micro-organisms
les s than 7 days.
V -A480 Appendix XVI F 2014
previously found or associated with the particular type of 2.6.12, and use 10 mL or the quantity corresponding to 1 g
cells. or 1 mL to inoculate a suitable amount (determined as
Other approaches to validation may also be used, for described under section 3-4 of general chapter 2.6.13) of
example, interlaboratory comparison. casein soya bean digest broth, mix and incubate at 30-35 oC
for 18-24 h.
TESTING OF THE PREPARATION TO BE EXAMINED
Sample A representative sample incJuding cells and/or Selection and sub culture Shake the container, transfer
medium is tested. The sample is added to the culture 1 mL of casein soya bean digest broth to 100 mL of
medium as soon as possible after collection. If it is not MacConkey broth and incubate at 42-44 oC for 24-48 h.
added promptly after collection, it is stored at 5 ± 3 oC to Subculture on a plate of MacConkey agar at 30-35 oC for
avoid phagocytosis of micro-organisms by ce lis present in 18-72 h.
certain types of products (for example, neutrophils). Interpretation Growth of colonies indica tes the possible
For haematopoietic products, the minimum amount to be presence of E. eali. This is confirmed by identification tests.
used for the test depending on the total volume of the The product complies with the test if no colonies are present
product (V mL) is shown below. or if the identification tests are negative.
Enumeration test Semi-quantitative test (probable
number method).
Total product volume Inoculum volume
(millilitres) Sample preparation and pre-incubation Prepare a
sample using a 1 in 10 dilution of not less than 1 g of the
v~ 10 1 per eent of total volume product to be examined as described in general chapter
l';V<lO
2.6.12, anduse the quantities corresponding respectively to
100l.1L
0.1 g, 0.01 g and 0.001 g (or 0.1 mL, 0.01 mL and
V< 1 Not applicable 0.001 mL) to inoculate a suitable amount (determined as
described under section 3-4 of general chapter 2.6.13) of
For haematopoietic products that require dilution before casein soya bean digest broth, mix and incubate at 30-35 oC
freezing, the inoculum volume must be increased by the for 18-24 h.
diJution factor. For other cellular products, suitable Selection and subculture Shake the container, transfer
minimum amounts are defined in terms of volume or 1 mL of casein soya bean digest broth to 100 mL of
number of doses. MacConkey broth and incubate at 42-44 oC for 24-48 h.
Analysis Samples are inoculated into containers of culture Subculture on a plate of MacConkey agar at 30-35 oC for
medium as soon as possible after collection and incubated at 18-72 h.
35-37 oC for not less than 7 or 14 days, depending on the Interpretation Growth of colonies indica tes the possible
detection system used. A suitable proportion of the inoculum presence of E. coli. This is confirmed by identification tests.
is added to the medium to be incubated in aerobic Note the smallest quantity of the product that gives a positive
conditions and the remainder of the inoculum to the result and the largest quantity that gives a negative result.
medium to be incubated in anaerobic conditions. Determine from the following table the probable number of
OBSERVATION AND INTERPRETATION OF RESULTS bacteria.
Examine media, visually or with automated systems at least
daily, and at the end of the observation period for evidence Probable
Results for each quantity of product
of microbial growth. If no growth is observed during or at number of
the end of the observation period, the product is 'culture bacteria per
0.01 g or 0.001 g or gram or
negative' at the limit of detection. If growth is observed in a 0.1 g or 0.1 mL
0.01 mL 0.001 mL millilitre of
valid test, the product is 'culture positive'; the contaminant is product
identified to a suitable taxonomic level (genus, species) and + + + > lO'
an antibiogram is established.
+ + < 10' and > lO'
+ < lO' and > 10
F. Microbiological Examination of Herbal <10
Medicinal Products for Oral Use

BILE-TOLERANT GRAM-NEGATIVE BACTERIA
Total aerobic microbial count (TAMC) Perform the Enumeration test Semi-quantitative test (probable
count as described in general chapter 2.6.12. number method).
Total combined yeasts/moulds count (TYMC) Perform Sample preparation and pre-incubation Prepare a
the count as described in general chapter 2.6.12. Due to the sample using a 1 in 10 dilution of not less than 1 g of the
natural high load of bacteria in the products covered by product to be examined as described in general chapter
general chapter 5.1.8, Sabouraud-dextrose agar containing 2.6.12, but using casein soya bean digest broth as the chosen
antibiotics may be used. diluent, mix and incubate at 20-25 oC for a time sufficient to
resuscitate the bacteria but not sufficient to encourage
SPECIFIED MICRO-ORGANISMS multiplication of the organisms (2 h to 3 h).
ESCHERICHIA COLl Selection and subculture Inoculate suitable quantities of
Test lar absenee enterobacteria enrichment broth-Mossel with the preparation
Sample preparation and pre-incubation Prepare a as described aboye and/or, depending on the limit applied
sample using a 1 in 10 dilution of not less than 1 g of the for the particular product, with 3 of the 4 dilutions of the
product to be examined as described in general chapter preparation, which contain respectively 0.1 g, 0.01 g,O.OOI
2014 Appendix XVI G V-A481
and 0.0001 g (or 0.1 mL, 0.01 mL, 0.001 and 0.0001 mL)
ofthe product to be examined. Incubate at 30-35 oC for
G. Microbiological Quality of Herbal
24-48 h. Subculture each of the cultures on a plate of violet Medicinal Products for Oral Use
red bile glucose agar. Incubate at 30-35 oC for 18-24 h .
(Ph. Eur. general text 5.1.8)
Interpretation Growth of colonies constitutes a positive This ehapter presems reeommended aeceptance eriteria for
result. Note the smallest quantity of the product that gives a microbiologieal quality of herbal medicinal products.
positive result and the largest quantity that gives a negative
The presence of certain micro-organisms in non-sterile
result.
preparations may have the potential to reduce or even
Determine from the following table the probable number of inactivate the therapeutic activity of the product and has the
bacteria. potential to adversely afIect the health of the patient.
Manufacturers have therefore 10 ensure a low bioburden of
ResuIts íor each quantily oC product Probable finished dosage forms by implementing current guidelines on
number oí
bacteria
Good Manufactunng Practice during the manufacture,
0.1 g or 0.01 g or 0.001 g or 0.0001 g or per gram or storage and distribution of pharmaceutical preparations.
0.1 mL 0.01 mL 0.001 mL 0.0001 mL millilitre oí
product
Microbial examination of non-stenle products is performed
according 10 the methods given in general chapters 2.6.12,
+ + + + > lO'
2.6.13 and 2.6.31. Acceptance cnteria for non-stenle
+ + + < la' and pharmaceutical products based upon the total aerobic
> lO'
microbial count (TAMC) and the total combined
+ < 103 and
+ yeasts/moulds count (TYMC) are given below.
> lO'
+ < lO' and Acceptance cnteria are based on individual results or on the
>10 average of replicate counts when replicate counts are
<10 performed (e.g. direct plating methods).
A list of specified micro-organisms for which acceptance
SALMONELLA entena are set can be found below. The list is not necessanly
exhaustive and for a given preparation it may be necessary 10
Test for absence
test for other micro-organisms depending on the nature of
Sample preparation and pre-incubation Prepare the
the starting materials and the manufactunng process.
2.6.12 and use the quantity corresponding to not les s than A. Herbal medicinal products containing herbal
25 g or 25 mL of the product 10 inoculate 225 mL of drugs, with or without excipients, intended for the
buffered pep10ne medium and mix (e.g. homogenise in a preparation of infusions and decoctions using
stomacher filter bag by using a blender) . Incubate at boiling water (for example herbal teas, with or
30-35 oC for 18-24 h. without added flavourings)
Buffered peptone medium
TAMC (2.6.12) Aeceptanee criterian: lO' CFU/ g
Potassium dihydrogen phosphate 1.5 g
Maximum aeeeptable eaunt: 50 000 000 CFU/ g
Disodium hydrogen phosphate dodecahydrate 9.0 g
TYMC (2.6.12) Aeeeptanee eriterian: lO' CFU/ g
Maximum aeeeptable eaunt: 500 000 CFU/ g
Peptone 10.0 g
Escherichia coli
Purified water 1000 mL Aeeeptance criterian : 103 CFU/ g
(2.6.31)
Adjust the pH so that after sterilisation it is 7.0 ± 0.2 at Salmonella (2.6.31) Absenee (25 g)
25 oc. Sterilise in an autoclave using a validated cycle.
Selection and subculture Transfer 0.1 mL of buffered B. Herbal medicinal products containing, for
peptone medium to 10 mL of Rappaport Vassiliadis example, extracts andlor herbal drugs, with or
Salmonella enrichment broth and incubate at 30-35 oC for without excipients, where the method of processing
18-24 h . Sub culture on plates of xylose, Iysine and (for example, extraction) or, where appropriate, in
deoxycholate agar, Incubate at 30-35 oC for 18-48 h. the case ofherbal drugs, ofpre-treatment reduces
Interpretation The possible presence of Salmonella is the levels of organisms to below those stated for
indicated by the growth of well-developed, red colonies, with this category
or without black centres . This is confirmed by identification
tests . TAMC (2.6.12) Aceeptanee criterion : lO' CFU/ g or CFU/ mL
The product complies with the test if colonies of the types Maximum aeeeptable eount : 50 000 CFU/ g
described are not present or if the identification tests are or CFU/ mL
negative. TYMC (2.6.12) Aeeeptance eriterian: lO' CFU/ g or CFU/ mL
Maximum aceeptable count: 500 CFU/ g or CFU/ mL
The recommended solutions and media are described in
Bile·tolerant
general chapter 2.6.13. gram·negative Acceptance eriterion: lO' CFU/ g ar CFU/ mL
bacteria (2.6.31)
Escherichia coli
Absence (1 g ar 1 mL)
(2.6.31)
Salmonella (2.6.31) Absenee (25 g ar 25 mL)
V-A482 Appendix XVI G 2014
C. Herbal medicinal products containing, for

example, extracts andlor herbal drugs, with or
without excipients, where it can be demonstrated
that the method of processing (for example,
extraction with low strength ethanol or water that
is not boiling or low temperature concentration)
or, in the case ofherbal drugs, ofpre-treatment,
would not reduce the level of organisms sufficiently
to reach the criteria required under B
TAMC (2.6.12) Acceptance criterion: 105 CFU/ g or CFU/ rnL

Maxirnurn acceptable count: 500 000 CFU/ g
or CFU/ rnL
TYMC (2.6.12) Acceptance criterion: 10 4 CFU/ g or CFU/ rnL
Maxirnum acceptable count: 50 000 CFU/ g
or CFU/ rnL
Bile-tolerant
grarn-negative Acceptance criterion: 10 4 CFU/ g or CFU/rnL
bacteria (2.6.31)
Escherichia coli
Absence (1 g or 1 rnL)
(2.6.31)
Salmonella (2.6.31) Absence (25 g or 25 mL)
lt is recognised that for some herbal medicinal produces the criteria

given above under A, B or C for TAMC, TYMC and bile-
tolerant gram-negative bacteria cannot be met because of the
typical level of microbial contamination. Higher acceptance criteria
may be applied on the basis of a risk assessment that takes
account of qualitative and quantitative characterisation of the
bioburden and the intended use of the medicinal product.
If it has been shown thar none of the prescribed tests will
allow valid enumeration of micro-organisms at the level
prescribed, a validated method with a limit of detection as
close as possible to the indicated acceptance criterion is used.
2014 Appendix XVII B V -A483
be validated. Light diffraction is also a widely used technique

Appendix XVII for measuring the size of a wide range of partic1es.
Where the cumulative distribution has been determined by
analytical sieving or by application of other methods, partic1e
A. Particle Size of Powders size may be characterised in the following manner:
1. Partic1e size c1assification of powders XgO partic1e size corresponding to 90 per cent of the
(Ph. Eur. method 2.9.12, Sieve test) cumulative undersize distribution;
The degree of fineness of a powder may be expressed by Xso median partic1e size (i.e. 50 per cent of the partic1es
reference to sieves that comply with the specifications for are smaller and 50 per cent of the partic1es are
non-analytical sieves (2.1.4). larger);
X¡O = partic1e size corresponding to 10 per cent of the
Where the degree of fineness of powders is determined by cumulative undersize distribution.
sieving, it is defined in relation to the sieve number(s) used
either by means of the following terms or, where such terms It is recognised that the symbol d is also widely used to
cannot be used, by expressing the fineness of the powder as a designate these values. Therefore, the symbols dgo , dso , dIO
percentage m/m passing the sieve(s) used. may be used.
The following terms are used in the description of powders: The following parameters may be defined based on the
Coarse powder Not less than 95 per cent by mas s passes cumulative distribution.
through a number 1400 sieve and not more than 40 per cent Q r(x) = cumulative distribution of partic1es with a dimension
by mass passes through a number 355 sieve. less than or equal to x where the subscript r reflects the
Moderately fine powder Not less than 95 per cent by distribution type.
mass passes through a number 355 sieve and not more than
r Dislribution type
40 per cent by mass passes through a number 180 sieve.
Fine powder Not less than 95 per cent by mass passes O Num ber
through a number 180 sieve and not more than 40 per cent 1 Length
by mass passes through a number 125 sieve.
2 Area
Very fine powder Not less than 95 per cent by mass
passes through a number 125 sieve and not more than 3 Volume
40 per cent by mass passes through a number 90 sieve.
If a single sieve number is given, not less than 97 per cent of Q.(X) = 0.90 when x = XgO
the powder passes through the sieve of that number, unless Q r(x) = 0.50 when x = Xso
otherwise prescribed.
Q.(x) = 0.10 when x = x¡o
Assemble the sieves and operate in a suitable manner until
An alternative but les s informative method of classifying
sifting is practically complete. Weigh the separated fractions
powder fineness is by use of the descriptive terms in
of the powder.
Table 2.9.35.-1.
Additional points for monographs other than those of
the European Pharmacopoeia Table 2.9.35.-1.
Within the monographs of the British Pharmacopoeia, the Classification oí powders by fineness
aboye terms may be used to specify the degree of coarseness
or fineness of a medicinal or pharmaceutical substance in Cumulative
Descriptive term X so (¡lm) distribution by volume
powder form that is to be incorporated into a formulated basis, Q3 (x)
preparation. The following terms may also be used for such
purposes. Coarse > 355 Q3(355) < 0.50
When the use of sieves is inappropriate, the definition is Q3(180) < 0.50 and
Moderately fine 180 - 355
expressed in terms of the partic1e size as determined by Q3(355) " 0.50
suitable microscopical examination.
Q3(l 25) < 0.50 and
Fine 125 - 180
Moderately coarse powder Not les s than 95% by weight Q3(180) " 0.50
passes through a number 710 sieve and not more than 40%
Very fine ,; 125 Q3(l25) " 0.50
by weight passes through a number 250 sieve.
Microfine powder Not less than 90% by weight passes
through a number 45 sieve.
Superfine powder Not less than 90% by number of the
partic1es are less than 1O ~lm in size. B. Sieves and Filters
2. Powder fineness 1. Sieves
(Ph. Eur. mechod 2.9.35) (Ph. Eur. mechod 2.1.4)
Partic1e-size distribution is estimated by analytical sieving Sieves are constructed of suitable materials with square
(2.9.38) or by application of other suitable methods where meshes. For purposes other than analytical procedures, sieves
appropriate. A simple descriptive classification of powder with circular meshes may be used, the internal diameters of
fineness is provided in this chapter. For practical reasons, which are 1.2 5 times the aperture of the square mesh of the
sieves are commonly used to measure powder fineness. corresponding sieve size. There must be no reaction between
Sieving is most suitable where a majority of the particles are the material of the sieve and the substance being sifted.
larger than about 75 ).lm, although it can be used for sorne Degree of comminution is prescribed in the monograph using
powders having smaller particle sizes where the method can the sieve number, which is the size of the mesh in
V -A484 Appendix XVII B 2014
Table 2.1.4.-1 (values in micrometers)

Sieve Tolerances Cor apertures Wire diameters
numbers
Maximum Tolerance Cor Intermediary Recommended Admissible limits
(Nominal
tolerance for mean aperture tolerance nominal
dimensions of
an aperlure dimensions
apertures)
+X :tY +Z d d~ dm1n
11200 770 350 560 2500 2900 2100
8000 600 250 430 2000 2300 1700
5600 470 180 320 1600 1900 1300

4000 370 130 250 1400 1700 1200
2800 290 90 190 U20 1300 950
2000 230 70 150 900 1040 770
1400 180 50 no 710 820 600
1000 140 30 90 560 640 480
710 112 25 69 450 520 380
500 89 18 54 315 360 270
355 72 13 43 224 260 190
250 58 9.9 34 160 190 130
180 47 7.6 27 125 150 106
125 38 5.8 22 90 104 77
90 32 4.6 18 63 72 54
63 26 3.7 15 45 52 38
45 22 3.1 13 32 37 27
38 - - - 30 35 24
micrometres, given in parenthesis after the name of the 2. Filters

substance (Table 2. 1.4.-1) (Ph. Eur. method 2.1.2)
Maximum tolerance 1 for an aperture (+ X): no aperture size Comparative table 01 porosity 01 sintered-glass jilters2
shall exceed the nominal size by more than X, where:
Table 2.1.2.-1
Porosity number Maximum Gennany France United
2 (W O.75 ) (Ph. Eur.)(3) di.meter of pores Kingdom
X = 3
+ 4 (W O. 25 ) in micrometres
1.6 less than 1.6 51
1- 2.5
w = width of aperture. 1.6·4
Tolerance for mean aperture (± Y): the average aperture 4·6

size shall not depart from the nominal size by more than 10 4 - 10 4f
± Y, where: 16 10 - 16
40 16 - 40
WO. 98
40 - 50
Y= - - +1.6
27 100 40 - 100
100 - 120
Intermediary tolerance (+ Z): not more than 6 per cent of
160 100 - 160
the total number of apertures shall have sizes between
150 - 200
" nominal + X" and " nominal + Z", where:
250 160·250
200 ·500 00
z= X+Y
2 Special Uses
Diameters in micrometres
Wire diameter d: the wire diameters given in Table 2.1.4 .-1 < 2.5 Bacteriological filtration
apply to woven metal wire cloth mounted in a frame . 4· 10 Ultra-fine filtration, separation of micro-organisms of large
The nominal sizes of the wire diameters may depart from diameter
10 ·40 Analytical filtration, very fine filtration of mercur)', very fine
these values within the limits dmax and dmin' The Jimits define dispersion of gases
a permissible range of choice ± 15 per cent of the 40· 100 Fine filtration, filtration of mercury, fine dispersion of gases
recommended nominal dimensions . The wires in a test sieve 100· 160 Filtration o( coarse materials, dispersion and washing of gases,
shall be of a similar diameter in warp and weft directions. support (or other filter materials
160·500 Filtration of very coarse materials, dispersion and washing of
gases.
2 The given limits are on/y approximate.

3 The European Phannacopoeia has adopted the system proposed by the
1 See che Imemarional Standard ISO 3310/1 (1975). Internalional Organisalion jor Standardisation (ISO).
2014 Appendix XVII B V-A485
3. Partic1e-size distribution estimation by analytical intended for use where at least 80 per cent of the particles
• • 1
sleVlng are larger than 75 )lm. The size parameter involved in
(Ph. Eur. method 2.9.38) determining particle-size distribution by analytical sieving is
Sieving is one of the oldest methods of classifying powders the length of the side of the minimum square aperture
and granules by particle-size distribution. When using a through which the particle will pass.
woven sieve cloth, the sieving will essentially sort the particles
by their intermediate size dimension (i.e. breadth or width). TEST SIEVES
Mechanieal sieving is most suitable where the majority of the Test sieves suitable for pharmacopoeial tests conform to the
particles are larger than about 75 )lm. For smaller particles, current edition of ISO 3310-1: Test sieves - Technical
their light weight provides insufficient force during sieving to requirements and testing - Part 1: Test sieves oi metal wire cloth
overcome the surface forces of cohesion and adhesion that (see Table 2 .9.38.- 1). Unless othelWise specified in the
cause the particles to stick to each other and to the sieve, and monograph, use those ISO sieves listed as principal sizes in
thus cause particles that would be expected to pass through Table 2.9.38.-1 that are recommended in the particular
the sieve to be retained. For such materials other means of region.
agitation such as air-jet sieving or sonie-sifter sieving may be
more appropriate. Nevertheless, sieving can sometimes be Table 2.9.38.-1.
used for sorne powders or granules having median particle
ISO Nominal Aperture US Recommend- European Japanese
sizes smaller than 75 )lm where the method can be validated. Sieve ed USP Sieve Sieve
In pharmaceutical terms, sieving is usually the method of Principal Supplementary No. Sieves (IJm) No. No.
sizes sizes
choice for classification of the coarser grades of single R20/3 R20 R40/3
powders or granules. It is a particularly anractive method in
11.20 mm 11.20 mm 11.20 mm 11 200
that powders and granules are classified only on the basis of
particle size, and in most cases the analysis can be carried 10.00 mm
out in the dty state. 9.50 mm
Among the limitations of the sieving method are the need for 9.00 mm
an appreciable amount of sample (normally at least 25 g,
8.00 mm 8.00 mm 8.00 mm
depending on the density of the powder or granule, and the
diameter of the test sieves) and the difficulty in sieving oily or 7.10 mm
other cohesive powders or granules that tend to clog the sieve 6.70 mm
openings. The method is essentially a two-dimensional 6.30 mm
estimate of size because passage through the sieve aperture is
5.60 mm 5.60 mm 5.60 mm 5600 3.5
frequently more dependent on maximum width and thickness
than on length. 5.00 mm
This method is intended for estimation of the total particle- 4.75 mm 4
size distribution of a single material. It is not intended for 4.50 mm
determination of the proportion of particles passing or
retained on 1 or 2 sieves. 4.00 mm 4.00 mm 4.00 mm 5 4000 4000 4.7
Estimate the particle-size distribution as described under Dry 3.55 mm

sieving method, unless otherwise specified in the individual 3.35 mm 6 5.5
monograph. Where difficulty is experienced in reaching the 3.15 mm
endpoint (i.e. material do es not readily pass through the
sieves) or when it is necessary to use the finer end of the 2.80 mm 2.80 mm 2.80 mm 7 2800 2800 6.5
sieving range (below 75 )lm), serious consideration must be 2.50 mm
given to the use of an altemative particle-sizing method. 2.36 mm 8 7.5
Sieving is carried out under conditions that do not cause the
2.24 mm
test sample to gain or lose moisture. The relative humidity of
the environment in which the sieving is carried out must be 2.00 mm 2.00 mm 2.00 mm 10 2000 2000 8.6
controlled to prevent moisture uptake or loss by the sample. 1.80 mm
In the absence of evidence to the contraty, analytical test 1.70 mm 12 lO
sieving is normally carried out at ambient humidity.
1.60 mm
Any special conditions that apply to a particular material
must be detailed in the individual monograph. 1.40 mm 1.40 mm 1.40 mm 14 1400 1400 12
PrincipIes of anaIytical sieving Analytical test sieves are 1.25 mm
constructed from a woven-wire mesh, which is of simple weave 1.18 mm 16 14
that is assumed to give nearly square apertures and is joined to
the base of an open cylindrical container. The basic analytieal 1.12 mm
method involves stacking the sieves on top of one another in 1.00 mm 1.00 mm 1.00 mm 18 1000 1000 16
ascending degrees of coarseness, and then placing the test 900 IJm
powder on the top sieve. The nest of sieves is subjected to a
850 IJm 20 18
standardised period of agitation, and then the mas s of material
retained on each sieve is accurately determined. The test gives 800 IJm
the mass percentage of powder in each sieve size range. 710 IJm 710 IJm 710 IJm 25 710 710 22
This sieving process for estimating the particle-size 630 IJm
distribution of a single pharmaceutical powder is generally
600 IJm 30 26
1 This chapter has undergone pharnzacopoeial hannonisation. See chapter
5.8. Phannacopoeial hannonisarion.
V-A486 Appendix XVII B 2014
ISO Nominal Aperture US Recommend· European Japanese sieve analysis at controlled room temperature and at ambient
Sieve ed USP Sieve Sieve relative humidity.
Principal Supplementary No. Sieves (J.lIII) No. No.
sizes sizes Cleaning test sieves Ideally, test sieves are cleaned using
R20/3 R20 R 40/3
only a low-pressure air jet or a liquid stream. If sorne
560 "m apertures remain blocked by test particles, careful gentle
500 "m 500 "m 500 "m 35 500 500 30 brushing may be used as a last resort.
450 "m
Test sample If the test sample mass is not given in the
monograph for a particular material, use a test sample having
425 "m 40 36
a mass of 25-100 g, depending on the bulk density of the
400 "m material, for test sieves having a 200 mm diameter.
355 "m 355 "m 355 ~m 45 355 355 42 For 76 mm sieves, the amount of material that can be
accommodated is approximately 1/7 that which can be
315 ~m
accommodated by a 200 mm sieve. Determine the most
300 ~m 50 50 appropriate mass for a given material by test sieving
280 ~m accurately weighed samples of different masses, such as 25 g,
50 g and 100 g, for the same time period on a mechanical
250 ~m 250 "m 250 ~m 60 250 250 60
shaker (note: if the test results are similar for the 25 g and
224 ~m 50 g samples, but the 100 g sample shows a lower
212 ~m 70 70 percentage through the finest sieve, the 100 g sample size is
200 ~m
too large) . Where only a sample of 10-25 gis available,
smaller diameter test sieves conforming to the same mesh
180 "m 180 ~m 180 ~m 80 180 180 83 specifications may be substituted, but the endpoint must be
160 ~m redetermined. The use of test samples having a smaller mas s
150 ~m 100 100 (e.g. down to 5 g) may be needed. For material s with low
apparent particle density, or for materials mainly comprising
140 ~m
particles with a high1y iso-diametrical shape, sample mas ses
125 ~m 125 ~m 125 ~m 120 125 125 119 below 5 g for a 200 mm screen may be necessary to avoid
112 ~m
excessive blocking of the sieve. During validation of a
particular sieve-analysis method, it is expected that the
106 ~m 140 140
problem of sieve blocking will have been addressed.
100~m
If the test material is prone to absorbing or losing significant
90 ~m 90 ~m 90 ~m 170 90 90 166 amounts of water with varying humidity, the test must be
80 ~m
.carried out in an appropriately controlled environment.
Similarly, if the test material is known to develop an
75 ~m 200 200
electrostatic charge, careful observation must be made to
71 ~m ensure that such charging do es not influence the analysis.
63 ~m 63 ~m 63 ~m 230 63 63 235 An antistatic agent, such as colloidal silicon dioxide andlor
aluminum oxide, may be added at a 0.5 per cent (m /m) level
56 ~m
to minimise this effect. If both of the aboye effects cannot be
53 ~m 270 282 eliminated, an alternative particle-sizing technique must be
50 ~m selected.
45 ~m 45 "m 45 ~m 325 45 45 330 Agitation methods Several different sieve and powder-
agitation devices are commercially available, all of which may
40 "m
be used to perform sieve analyses. However, the different
38 ~m 38 391 methods of agitation may give different results for sieve
analyses and endpoint deterrninations because of the
different types and magnitudes of the forces acting on the
Sieves are selected to cover the entire range of particle sizes individual particles under test. Methods using mechanical
present in the test sample- A nest of sieves having a V2 agitation or electromagnetic agitation, and that can induce
progression of the area of the sieve openings is either a vertical oscillation or a horizontal circular motion, or
recommended. The nest of sieves is assembled with the tapping or a combination of both tapping and horizontal
coarsest screen at the top and the finest at the bottom. circular motion are available. Entrainment of the particles in
U se micrometres or millimetres in denoting test sieve an air stream may also be used. The results must indicate
openings. which agitation method was used and the agitation
Test sieves are made from stainless steel or, less preferably, parameters used (if they can be varied), since changes in the
from brass or another suitable non-reactive wire. agitation conditions will give different results for the sieve
Calibration and recalibration of test sieves is in accordance analysis and endpoint determination, and may be sufficiently
with the current edition ofISO 3310-1. Sieves are carefully different to give a failing result under sorne circumstances.
examined for gross distortions and fractures, especially at Endpoint determination The test sieving analysis is
their screen frame joints, before use. Sieves may be calibrated complete when the mass on any of the test sieves do es not
optically to estimate the average opening size, and opening change by more than 5 per cent or 0.1 g (lO per cent in the
variabiliry, of the sieve mesh. Alternatively, for the evaluation case of 76 mm sieves) of the previous mass on that sieve.
of the effective opening of test sieves in the size range of If les s than 5 per cent of the total sample mas s is present on
212-850 ¡.tm, standard glass spheres are available. Unless a given sieve, the endpoint for that sieve is increased to a
otherwise specified in the individual monograph, perforrn the
2014 Appendix XVII e V-A487
mass change of not more than 20 per cent of the previous is very important to confirm that the oversize material
mass on that sieve. comprises single particIes and is not composed of aggregates.
If more than 50 per cent of the total sample mass is found
INTERPRETATION
on any one sieve, unless this is indicated in the monograph,
the test is repeated, but with the addition to the sieve nest of The raw data must incIude the mas s of the test sample, the
a more coarse sieve intermedia te between that carrying the total sieving time, the precise sieving methodology, and the
excessive mass and the next coarsest sieve in the original set values for any variable parameters, in addition to the
nest, i.e. addition of the ISO series sieve omitted from the mas ses retained on the individual sieves and in the pan.
nest of sieves. It may be convenient to convert the raw data into a
cumulative mas s distribution, and if it is desired to express
SIEVING METHODS the distribution in terms of a cumulative mass undersize, the
Mechanical agitation (Dry sieving method) Tare each range of sieves used must include a sieve through which all
test sieve to the nearest 0.1 g. Place an accurately weighed the material passes. If there is evidence on any of the test
quantity of test sample on the top (coarsest) sieve, and sieves that the material remaining on it is composed of
replace the lid. Agitate the nest of sieves for 5 min, then aggregates formed during the sieving process, the analysis is
carefully remove each sieve from the nest without loss of invalido
material. Reweigh each sieve, and determine the mass of
material on each one. Determine the mass of material in the
collecting pan in a similar manner. Re-assemble the nest of C. Specific Surface Area by Air
sieves, and agitate for 5 mino Remove and weigh each sieve
as previously described . Repeat these steps until the endpoint Permeability
criteria are met (see Endpoint determination under Test (Ph. Eur. methad 2.9.14)
sieves). Upon completion of the analysis, reconcile the The test is intended for the determination of the specific
masses of material. Total loss must not exceed 5 per cent of surface area of dry powders expressed in square metres per
the mass of the original test sample. gram in the sub-sieve region. The effect of molecular ftow
Repeat the analysis with a fresh sample, but using a single ("slip ftow ") which may be important when testing powders
sieving time equal to that of the combined times used aboye. consisting of particIes less than a few micrometres is not
Confirm that this sieving time conforms to the requirements taken into account in the equation used 10 calcula te the
for endpoint determination. When this endpoint has been specific surface area.
validated for a specific material, then a single fixed time of
sieving may be used for future analyses, providing the Apparatus
particle-size distribution falls within normal variation. The apparatus consists of the following parts:
If there is evidence that the particles retained on any sieve are (a) a penneability cell (see Figure 2.9.14.-1 ), which consists
aggregates rather than single particles, the use of mechanical of a cylinder with an inner diameter of 12.6 ± 0.1 mm
dry sieving is unlikely to give good reproducibility, and a (A), constructed of glass or non-corroding metal.
different particIe-size analysis method must be used. The bottom of the cell forms an airtight connection (for
Air-entrainment methods (Air-jet and sonic-sifter example, via an adapter) with the manometer (Figure
sieving) Different types of commercial equipment that use 2.9.14.-2). A ledge 0.5 mm to 1 mm in width is located
a moving air current are available for sieving. A system that 50 ± 15 mm from the top of the cell. It is an integral
uses a single sieve at a time is referred to as air-jet sieving. part of the cell or firmly fixed so as to be airtight.
It uses the same general sieving methodology as that It supports a perforated metal disk (E), constructed of
described under Dry sieving method, but with a standardised non-corroding metal. The disk has a thickness of
air jet replacing the normal agitation mechanism. It requires 0.9 ± 0.1 mm and is perforated with thirty 10 forty
sequential analyses on individual sieves starting with the holes 1 mm in diameter evenly distributed over this area.
finest sieve to obtain a particIe-size distribution. Air-jet The plunger (C) is made of non-corroding metal and fits
sieving often includes the use of finer test sieves than used in into the cell with a clearance of not more than 0. 1 mm.
ordinary dry sieving. This technique is more suitable where The bottom of the plunger has sharp square edges at
only oversize or undersize fractions are needed. right angles to the principal axis. There is an air vent
3 mm long and 0.3 mm deep on one side of the
In the sonic-sifter method, a nest of sieves is used, and the
plunger. The top of the plunger has a collar such that
test sample is carried in a vertically oscillating column of air
when the plunger is placed in the cell and the collar is
that lifts the sample and then carries it back against the mesh
brought into contact with the top of the cell, the
openings at a given number of pulses per minute. It may be
distance between the bottom of the plunger and the top
necessary to lower the sample amount to 5 g when sonic
of the perforated disk (E) is 15 ± 1 mm. The filter
sifting is employed.
paper disks (D ) have smooth edges and the same
The air-jet sieving and sonic-sifter sieving methods may be diameter as the inside of the cell.
useful for powders or granules when the mechanical sieving
(b) a U-tube manameter (E) (Figure 2.9.14.-2) is made of
techniques are incapable of giving a meaningful analysis.
nominal 9 mm outer diameter and 7 mm inner diameter
These methods are highly dependent upon proper dispersion glass tubing with standard walls. The top of one arm of
of the powder in the air current. This requirement may be the manometer forms an airtight connection with the
hard to achieve if the method is used at the lower end of the permeability cell (P). The manometer arm connected 10
sieving range (i.e. below 75 flm), when the particles tend to the permeability cell has a line etched around the tube at
be more cohesive, and especially if there is any tendency for 125 mm 10 145 mm below the lOp of the side outlet and
the material to develop an electrostatic charge. For the aboye three other lines at distances of 15 mm, 70 mm and
reasons endpoint determination is particularly critical, and it 110 mm aboye that line (G). The side outlet 250 mm to
305 mm aboye the bottom of the manometer is used 10
V -A488 Appendix XVII e 2014
evacuate the manometer arm connected to the quantity of the powder used. If, on the contrary, there is not
permeability cell. A tap is provided on the side oudet enough resistance, increase the quantity of the powder.
not more than 50 mm from the manometer armo In this case calculate the porosity again. After at least lOs,
The manometer is mounted firmly in such a manner that the remove the plunger.
arms are vertical. It is filled to the lowest mark with dibutyl
phthalate R containing a lipophilic dye. (F)
016
I
012.6 ± 0.1
~ I
I
-
I r
A) 1()
<O
I ,
1()
o
(B) I '" <O
. .:.1... 1 1()
N
(D)
~
., '1 ,"
~ I:Y.I.. .
"
~
¡~
I
I en
'"
c:::i
I (B)
I Figure 2.9.14.-2. - Manometer

I
Figure 2.9.14.-1. - Permeability cell Anach the permeability cell to the tube of the manometer by
means of an airtight connection. Evacuate the air from the
Dimensions in millimetres manometer by means of a rubber bulb until the level of the
coloured liquid is at the highest mark. Close the tap and
check that the apparatus is airtight by c10sing the upper end
Method
of the cell, for example with a rubber stopper. Remove the
If prescribed, dry the powder to be examined and sift stopper and, using a timer, measure the time taken for the
through a suitable sieve (for example no. 125) to disperse liquid to fall from the second to the third mark.
agglomerates. Calculate the mas s (M) of the powder to be
Using the measured flow time, calculate the specific surface
used from the following expression:
area (S), expressed in square metres per gram, from the
following expression:
M = Vxpx(l-é) (1)
V bulk volume of the compacted bed of powder,

s= KxVe3 x .,fi
(2)
p x (1 - é) X ..fo
p density of the substance to be examined in grams per
millilitre,
flow time in seconds,
e porosity of the compacted bed of powder.
dynamic viscosity of air in millipascal seconds (see
Assume first a porosity of 0.5 and introduce this value in Eq. Table 2.9 .14.-1),
1 to calculate the mass (M) of the powder to be examined. K apparatus constant determined according to Equation
Place a filter paper disk on top of the perforated metal disk (4),
(B). Weigh the calculated mass (M) of the powder to be p density of the substance to be examined in grams per
examined to the nearest 1 mg. Carefully transfer the powder millilitre,
into the c1eaned, tared permeability cell and carefully tap the porosity of the compacted bed of powder.
cell so that the surface of the powder bed is level and cover it
with a second filter paper disk. Slowly compact the powder
by means of the plunger, avoiding rotary movement.
Maintain the pressure until the plunger is completely inserted
into the permeability cell. If this is not possible, decrease the
2014 Appendix XVII E V-A489
Calibration of the apparatus Table 2.9. 14.-l.

Temperature Density of mercury Viscosity of air (11) .¡Tí
The bulk volume of the compacted bed of powder is (OC) (g¿mL) (mPa-s)
detennined by the mercury displacement method as follows:
16 13.56 0.01800 0.1342
Place two filter paper disks in the permeability cell, pressing
17 13.56 0.01805 0.1344
down the edges with a rod slightly smaller than the cell
diameter until the filter disks lie flat on the perforated metal 18 13.55 0.01810 0.1345
disk; fill the cell with mercury, removing any air bubbles 19 13.55 0.01815 0.1347
adhering to the wall of the cell and wipe away the excess 10
20 13.55 0.01819 0.1349
create aplane surface of mercury at the 10p of the cell. If the
cell is made of material that will amalgama te, grease the cell 21 13.54 0.01824 0.1351
and the metal disk first with a thin layer of liquid paraffin. 22 13.54 0.01829 0.1353
Pour out the mercury into a tared beaker and detennine the
mass (MA ) and the temperature of the mercury. 23 13.54 0.01834 0.1354
Make a compacted bed using the reference powder and again 24 13.54 0.01839 0.1356
fill the cell with mercury with aplanar surface at the top of
the cell. Pour out the mercury in a tared beaker and again
detennine the mass of the mercury (MB ). Calculate the bulk
volume (V) of the compacted bed of powder from the
following expression: E. Flowability
(Ph . Eur. mechad 2.9.16)
(3) The test for flowability is intended to determine the ability of
divided solids (for example, powders and granules) 10 flow
vertically under defined conditions.
difference between the detennined mas ses of
mercury in grams, Apparatus
PHg density of mercury at the detennined According 10 the flow properties of the material to be tested,
temperature in grams per millilitre. funnels with or without stem, with different angles and orífice
Repeat the procedure twice, changing the powder each time; diameters are used. Typical apparatuses are shown in Figures
the range of values for the calculated volume (V) is not 2.9.16.-1 and 2.9.16.-2. The funnel is maintained upright by
greater than 0.01 mL. Use the mean value of.the three a suitable device. The assembly must be protected from
detennined volumes for the calculations. vibrations.
The apparatus constant K is determined using a reference
powder with known specific surface are a and density as Method
follows: Into a dry funnel, whose bottom opening has been blocked
Calculate the required quantity of the reference powder 10 be by suitable means, introduce without compacting a test
used (Eq. 1) using the stated density and the detennined sample weighed with 0.5 per cent accuracy. The amount of
volume (lf the compacted powder bed (Eq. 3). the sample depends on the apparent volume and the
apparatus used. Unblock the bottom opening of the funnel
Homogenise and loo sen up the powder by shaking it for
and measure the time needed for the entire sample 10 flow
2 min in alOa mL bottle. Prepare a compacted powder bed
out of the funnel. Carry out three detenninations.
and measure the flow time of air as previously descríbed.
Calculate the apparatus constant (K) from the following Expression of results
expression:
The flowability is expressed in seconds and tenths of
seconds, related 10 100 g of sample.
K = _S....:.sp_x_p-,oX=('--l_-_€-=)_X----'-,fi1_T7 (4) The resuIts depend on the s10rage conditions of the material
..j¡3 x .Ji 10 be tested.
The results can be expressed as the following:
SSP stated specific surface area of the reference powder,
a) the mean of the detenninations, if none of the individual
P density of the substance to be examined in grams per
values deviates from the mean value by more than
millili tre,
la per cent;
porosity of the compacted bed of powder,
flow time in seconds, b) as a range, if the individual values deviate from the mean
IJ dynamic viscosity of air in millipascal seconds (se e value by more than la per cent;
Table 2.9.14 .-1). e) as a plot of the mass against the flow time;
The density of mercury and the viscosity of air over a range d) as an infinite time, if the entire sample fails to flow
oftemperatures are shown in Table 2.9.14.-1. through.
V-A490 Appendix XVII F 2014
0110
F. Measurement of Consistency and
Texture Analysis
1. Measurement of Consistency by Penetrometry

This test is intended to measure, under determined and
vaJidated conditions, the penetration of an object into the
product to be examined in a container with a specified shape
Connecting nut
and size.
Apparatus
The apparatus consists of a penetrometer made up of a stand
and a penetrating object. A suitable apparatus is shown in
Figure 2.9.9 .-1.
030.2 .g,
Nozzle 1, 2 or 3
enlarged
-d
Nozzle Diameter (á) oC the outflow __ ~ ______________ A

openinl! (millimetres)
1 10 ± 0.01
2 15 ± 0.01
3 25 ± 0.01
l~-------------------- B
Figure 2.9.16.-1. - Flow funnel and nozzle. Nozzle is made of
stainless, acid-resistant steel (V4A, CrNi) ~ _______________ C
_____________________ D
~~~------------- E
--+---~~------- F
__--'~---- G
Figure 2.9.9.-1. - Penetrometer
A. Scale showing the depth of penetration, graduated in

tenths of millimetres.
1.25___-+--1- B. Vertical shaft to maintain and guide the penetrating
object.
45° C. Device to retain and to release the penetrating object
automatically and for a constant time.
D. Device to ensure that the penetrating object is vertical
Figure 2.9.16.-2 and that the base is horizontal.
Dimensions in millimetres E. Penetrating object (see Figures 2.9 .9.-2 and 3) .
F. Container.
G. Horizontal base.
H. Control for the horizontal base.
2014 Appendix XVII F V -A491
65 ± 0 .25.
3.14 ± 0.04.
IX/<II------A
0d
~~----~~~
+1
a>
~~------~~~-------L
Figure 2.9.9.-2. - Cone (m = 102.5 ± 0.05 g), suitable container (d = 102 mm or 75 mm; h ~ 62 mm)
and shaft (1 = 162 mm; m = 47.5 ± 0.05 g).
012.65
09.5
i
03.2
08.2 -.. +-
r
r-.
7h o
~
r-.
l()
l()
C'?
CD
Figure 2.9.9.-3 - Micro-cone (m = 7.0 g), suitable container and shaft (1 =116 mm; m =16.8 g)
The stand is made up of: The penetrating object, made of a suitable material, has a
- a vertical shaft to maintain and guide the penetrating smooth surface, and is characterised by its shape, size and
object; mass (m).
- a horizontal base; Suitable penetrating objects are shown in Figures 2.9.9.-2
- a device to ensure that the penetrating object is vertical; and 2.9.9 .-3.
- a device to check that the base is horizontal; Procedure
- a device to retain and release the penetrating object; Prepare the test samples according to one of the following
- a scale showing the depth of penetration, graduated in procedures.
tenths of a millimetre. A. Carefully and completely fill 3 containers, without
forming air bubbles. Leve! if necessary to obtain a
V-A492 Appendix XVII F 2014
Travel ling arm moves probe up or dovvn
Probe
Sample stage
Fig. 17F-l Texture Analyser
fiat surface. Store the samples at 25 ± 0.5 oC for 24 h, This test applies to samples consisting of a semi solid or gel-
unless otherwise prescribed. like mass which retains its formo It is not applicable to
B. Store 3 samples at 25 ± 0.5 oC for 24 h, unless suspensions consisting of fine solid particles in a liquido
otherwise prescribed. Apply a suitable shear to the Apparatus A suitable texture analyser, (see Fig. 17F-l)
samples for 5 mino Carefully and completely fill 3 consisting of:
containers,' without forming air bubbles, and level if - a suitable platform or device to hold the sample being
necessary to obtain a fiat surface. examined,
C. Melt 3 samples and carefully and completely fill 3 - a mobile arm that can be moved in a vertical direction
containers, without forming air bubbles. Store the towards or away from the sample,
samples at 25 ± 0.5 oC for 24 h, unless otherwise
- a probe attachment, which may be ofvarious shapes such
prescribed.
as a platen, a cylinder, a cone, a needle, a sphere or a
Determination of penetration Place the test sample on wire,
the base of the penetrometer. Verify that its surface is
- a load cell capable of measuring the load or tensile force s
perpendicular to the vertical axis of the penetrating object.
experienced by the probe as the mobile arm moves up or
Bring the temperature of the penetrating object to
down.
25 ± 0.5 oC and then adjust its position such that its tip
just touches the surface of the sample. Release the Calibration The apparatus is calibrated using a suitable
penetrating object and hold it free for 5 S. Clamp the certified weight.
penetrating object and measure the depth of penetration. Method Check that the apparatus is vertical.
Repeat the test with the 2 remaining containers. Place the sample being examined in a suitable holder as
specified in the monograph. Programme the apparatus in
Expression of the results
accordance with the manufacturer's specifications to move
The penetration is expressed in tenths of a millimetre as the the probe up or down at a defined speed or force as specified
arithmetic mean of the 3 measurements. If any of the in the monograph. Measure the force s experienced by the
individual results differ from the mean by more than probe through the sample.
3 per cent, repeat the test and express the results of the 6 The maximum peak force (in g) required to penetrate the
measurements as the mean and the relative standard sample is measured by the load cel!.
deviation.
Carry out each measurement six times.
2. Texture Analysis of Semi-solids or Gels
(No Ph. Eur. methad)
This test determines, under defined conditions, the force
required to penetrate a semi-solid or gel sample.
2014 Appendix XVII G V-A493
specifications as far as friability is concerned. In the case of

G. Friability hygroscopic tablets, a humidity-controlIed environment is
required for testing.
1. Uncoated Tablets l
A drum with dual scooping projections, or apparatus with
more than one drum, for the running of multiple samples at
This chapter provides guidelines for the friability
one time, are also pennitted.
detennination of compressed, uncoated tablets. The test
procedure presented in this chapter is generally applicable to 2. Granules and Spheroids
most compres sed tablets. Measurement of tablet friability
supplements other physical strength measurements, such as (Ph. Eur. method 2.9.41)
tablet breaking force . This chapter describes 2 methods for determination of the friability
of granules and spheroids, which may be used during development
Use a drum, with an internal diameter between 283-291 mm
studies. It is recognised, however, that many methods with equal
and a depth between 36-40 mm, of transparent synthetic
suitability may be used.
polymer with polished internal surfaces, and subject to
minimum static build-up (see Figure 2.9.7.-1.). One side of This test is intended to detennine, under defined conditions,
the drum is removable. The tablets are tumbled at each turn the friability of granules and spheroids. Friability is defined as
of the drum by a curved projection with an inside radius a reduction in the mas s of the granules or spheroids or in the
between 75.5-85.5 mm that extends from the middle of the formation of fragments of granules or spheroids, occurring
drum to the outer wall. The outer diameter of the central when the granules or spheroids are subjected to mechanical
ring is between 24.5-25.5 mm. The drum is attached to the strain during handling (tumbling, vibration, fluidisation, etc .).
horizontal axis of a device that rotates at 25 ± 1 r/min. Examples of changes are abrasion, breakage or defonnation
Thus, at each turn the tablets roll or slide and fall onto the of granules or spheroids.
drum waIl or onto each other.
METHODA
Apparatus (fluidised-bed apparatus) The apparatus
(see Figure 2.9.41.-1) consists of a glass cylinder (A) with a
conical lower part. The cylinder is provided with a sieve lid
(B) having an aperture size of 500 ¡.tm or any other suitable
sieve. The conical end is connected to a U-shaped glass tube
(C) that can be disconnected from the cylinder for removal
of the granules or spheroids. The U-tube is attached to a
T-coupling (D). One inlet of the T-coupling is joined by a
silicone tube to a manometer for regulating the compressed-
air flow (use compres sed air complying with the test for
water in the monograph Me.dicinal air (1238), the other one
is connected via a silicone tube to a by-pass flowmeter (E)
(0.10-1.00 m 3 ·h- I ) .
38.0 :t2.0 mm
Procedure The following procedure is usually suitable.
Remove the fine particles by sieving (sieve having an
Figure 2.9.7.-1. - Tablet friability apparatus aperture size of 710 ¡.tm or any other suitable sieve).
Introduce about 8.0 g (mI) of granules or spheroids into the
For tablets with a unit mass equal to or less than 650 mg, cylinder (A) . Close the apparatus with the sieve lid (B) .
take a sample of whole tab1ets corresponding as near as Adjust the flow rate of the compressed air to 0.45 m 3 ·h - 1 .
possible to 6.5 g. For tablets with a unit mass of more than After 15 min, remove the granules or spheroids from the
650 mg, take a sample of 10 whole tablets . The tablets are apparatus by disconnecting the U-tube and weigh again
(m2)' Test 3 samples and calculate the mean value. It is
carefuIly dedusted prior to testing. Accurately weigh the
tablet sample, and place the tablets in the drum. Rotate the recommended to spray the inside of the apparatus with an
drum 100 times, and remove the tablets. Remove any 100se antistatic agent every 3 detenninations in order to prevent
dust from the tablets as before, and accurately weigh. electrostatic charging.
Loss ondrying Dry in an oven at 105 oC, unless
Generally, the test is run once. If obviously cracked, c1eaved,
otherwise prescribed. AIternatively, other drying conditions
or broken tablets are present in the tablet sample after
tumbling, the sample fails the test. If the resuIts are difficult as described in general chapter 2. 2.32 may be used.
to interpret or if the weight loss is greater than the targeted Calculation:
value, the test is repeated twice and the mean of the 3 tests
detennined. A maximum loss of mass (obtained from a single F = m¡ (100 - TI) - m2 (100 - T2) x 100
test or from the mean of 3 tests) not greater than m¡
1.0 per cent is considered acceptable for most products.
If tablet size or shape causes irregular tumbling, adjust the F friabilityi
drum base so that the base forms an angle of about 10 with 0
TI percentage loss on drying before the test (mean of 2
the horiZontal and the tablets no longer bind together when detenninations)i
lying next to each other, which prevents them from faIling T2 percentage loss on drying after the test (mean of 2
freeIy. detenninations) i
Effervescent tablets and chewable tablets may have different mI mass of the granules or spheroids before the test, in
gramsi
1 This chapter has undergone phannacopoeial hamzonisation. See chapter
mass of the granules or spheroids after the test, in
5.8 Pannacopoeial hamzonisation. grams.
V-A494 Appendix XVII G 2014
E
u
\O
A ..,:
~
E 5
\O
D
4.5 cm 5cm
E
V --++---=-1 cm
u
o
E
u
": E
o u
\O
Figure 2.9.41.-1. - Fluidised-bed apparatus
D
Glass container
TIme Frequency
107 ± 3 rnl glass container,
42.0 ± 0.5 mm in internal diameter
and 85.0 ± 1 mm in height,
with a twist-off cap.
Figure 2.9.41.-2. - Oscillating apparatus
METHODB Procedure The following procedure is usually suitable.

Apparatus (oscillating apparatus) The appararus (se e Remove the fine partic1es by sieving (sieve having an
Figure 2.9.41.-2) consists of a glass container, containing the aperture size of 355 ¡.tm or any other suitab1e sieve). In the
granules or spheroids to be examined, which is subjected lO glass container, weigh about 10.00 g (mi) ofthe granules or
horizontal oscillations. The frequency and duration of the spheroids. Install the container in the apparatus. Shake for
oscillations can be varied continuously. The frequency can 240 s at the highest frequency for hard granules or
be adjusted, using a scale, lO a value in the range 0-400 spheroids, or for 120 s at a lower frequency
oscillations/min. The duration can be set lO a value in the (e.g. 140 oscillations/min) for soft granules or spheroids.
range 0-9999 s. Sieve (355 ¡.tm, or the same sieve as used previously) and
2014 Appendix XVII J V-A495
weigh the gr~nules or spheroids again (m2)' Test 3 samples cover having an opening 5.2 mm in diameter. The apparatus
and calculate the mean value. comprises a rod 5.0 mm in diameter which becomes wider
Loss on drying Dry in an oven at 105 oC, unless towards the lower end, reaching a diameter of 12 mm.
otherwise prescribed. Alternatively, other drying conditions A metal needle 2 mm in length and 1 mm in diameter is
as described in general chapter 2.2.32 may be used. fixed on the fiat underside.
Calculation The rod consists of 2 parts, a lower part made of plastic
material and an upper part made of plastic material or metal
F = mI (100 - TI) - m2 (100 - T 2 ) x 100 with a weight disk. The upper and lower parts are either
mI fitted together (manual version) or separate (automated
version). The weight of the entire rod is 30 ± 0.4 g.
F friability; The upper part of the rod carnes a sliding mark ringo When
T¡ percentage los s on drying before the test (mean of 2 the rod is introduced into the glass tube so that it touches
determinations) ; the bottom, the mark ring is adjusted to coincide with the
T2 percentage loss on drying after the test (mean of 2 upper level of the plastic cover.
determinations);
m¡ mas s of the granules or spheroids before the test, in
grams;
m2 mass of the granules or spheroids after the test, in
grams.
H. Resistance to Crushing of Tablets

This test is intended to determine, under defined conditions, ~I
the resistance to crushing of tablets, measured by the force
needed to disrupt them by crushing.
Apparatus
The apparatus consists of 2 jaws facing each other, one of
which moves towards the other. The fiat surfaces of the jaws
are perpendicular to the direction of movement.
The crushing surfaces of the jaws are fiat and larger than the
zone of contact with the tablet. The apparatus is calibrated
using a system with a precision of 1 newton.
Operating procedure
Place the tablet between the jaws, taking into account, where Figure 2.9:22.-1. - Apparatus A far measuring the saftening
applicable, the shape, the break-mark and the inscription; time af lipaphilic suppasitaries
for each measurement orient the tablet in the same way with Dimensians in millimetres
respect to the direction of application of the force. Carry out
the measurement on 10 tablets, taking care that al! fragments Method Place the glass tube containing 10 mL of water in
of tablets have been removed before each determination. a water-bath and equilibrate at 36.5 ± 0.5 oC, Fix the glass
This procedure doés not apply when fully automated equipment is tube vertical!y and immerse to a depth of at least 7 cm
used. below the surface but without touching the bottom of the
water-bath. Introduce a suppository, tip first, into the tube
Expression of results
fol!owed by the rod with the free gliding plastic cover into
Express the results as the mean, minimum and maximum ' the glass tube until the metal needle touches the fiat end of
values of the forces measured, al! expressed in newtons. the suppository. Put the cover on the tube (beginning of
Indicate the type of apparatus and, where applicable, the time measurement). Note the time which elapses until the
orientation of the tablets. rod sinks down to the bottom of the glass tube and the mark
ring reaches the upper level of the plastic cover.
Apparatus B
J. Softening Time Determination of The apparatus (see Figure 2.9 .22.-2) consists of a water-bath
Lipophilic Suppositories (E) into which an inner tube (A) is inserted and fixed with a
(Ph. Eur. method 2.9.22) stopper. The inner tube is closed by a stopper at the bottom.
The test is intended to determine, under defined conditions, The apparatus is fitted with a thermometer. 2 insets are
the time which elap'ses until a suppository maintained in available:
water softens to the extent that it no longer offers resistance - a glass rod (C1) in the form of a tube sealed at both ends,
when a defined weight is applied. carrying a rirn at its lower end weighed with lead shot,
which has a weight of 30 ± 0.4 g,
Apparatus A
- a penetration inset (C2) consisting of a rod (7.5 ± 0.1 g)
The apparatus (se e Figure 2.9.22.-1) consists of a glass tube in a tube which has an enlargement for the suppository,
15.5 mm in internal diameter with a fiat bottom and a length both made of stainless steel.
of about 140 mm. The tube is closed by a removable plastic
V-A496 Appendix XVII K 2014
Method Pour 5 mL ofwater at 36.5 ± 0.5 oC into the - a system capable of pressurising the test cell with the
inner tube CA), introduce a suppository with the tip measurement gas until a defined pressure (P) indicated by
downwards and onto that, place the inset (C1 or C2). Note a manometer;
the time which elapses between this moment and the - the system is connected to a source of measurement gas,
moment when the lower, rimmed end of the glass rod (Cl) preferably helium, unless another gas is specified.
or the steel rod (C2) reaches the narrowed pan of the inner The gas pycnometric density measurement is perforrned at a
glass tube. Melting or dissolution is then considered as temperature between 15 oC and 30 oC that does not vary by
complete. more than 2 oC during the course of measurement.
The apparatus is calibrated, which means that the volumes
Ve and Vr are deterrnined using a suitable calibration
::12.5rL standard whose volume is known to the nearest 0.001 cm 3 .
The procedure described below is followed in 2 runs, firstly
with an empty test cell, and secondly with the calibrarion
standard placed in the test cel!. The volumes Ve and Vr are
r1-'-1 calculated using the equarion for the sample volume (V,) ,
taking into account that V, is zero in the first runo
Figure 2.9.22.-2. - Apparatus B for measuring the softening

time of lipophilic suppositories
A valve;
expansion volume, in cubic centimetres;
cell volume, in cubic centimetres ;
K. Pycnometric Density of Solids sample volume, in cubic centimetres;
M manometer.
Gas pycnometric density is deterrnined by measuring the
volume occupied by a known mass of powder, which is Figure 2.9.23.-1. - Schematic diagram of a gas pycnometer
equivalent to the volume of gas displaced by the powder
using a gas displacement pycnometer. In gas pycnometric
density measurements, the volume deterrnined exeludes the Method
volume occupied by open pores; however, it ineludes the Volatile contaminants in the powder are removed by
volume occupied by sealed pores or pores inaccessible to the degassing the powder under a constant purge of helium prior
gas. to the measurement. Occasionally, powders may have to be
Usually, helium is used as a test gas due to its high diffusivity degassed under vacuum. Because volatiles may be evolved
into small open pores. If gases other than helium are used, during the measurement, weighing of the sample is carried
different values would be obtained, since the penetration of out after the pycnometric measurement of volume.
the gas is dependent on the size of the pore as well as the Weigh the test cell of the pycnometer and record the mass.
cross-sectional area of the gas molecules. Fill the test cell with a given mass of powder of the substance
The measured density is a volume-weighted average of the to be examined. Seal the test cell in the pycnometer. Record
densities of individual powder partieles. It is called the the system reference pressure (Pr) as indicated by the
partiele density, distinct from the true density of a solid or manometer while the valve that connects the expansion cell
the"bulk density of a powder. The density of solids is with the test cell is open. Close the valve to separate the
expressed in grams per cubic centimetre (g/cm 3), although expansion cell from the test cel!. Pressurise the test cell with
the International Unit is the kilogram per cubic meter the gas to an inirial pressure (P¡) and record the value
(1 g/cm 3 = 1000 kg/m3 ) . obtained. Open the valve to connect the expansion cell with
Apparatus the test cel!. Record the final pressure (PI)' Repeat the
measurement sequence for the same powder sample until
The apparatus (see Figure 2.9.23.-1) consists ofthe consecutive measurements of the sample volume (V,) agree
following: to within 0.2 per cent. Unload the test cell and measure the
- a sealed test cell, with empty cell volume Ve , connected final powder mas s (m), expressed in grams. If the
through a valve to an expansion cell, with volume Vr ; pycnometer differs in operation or construction from the one
2014 Appendix XVII M V-A497
shown in Figure 2.9.23.-1, follow the instructions ofthe Then the BET value
manufacturer of the pycnometer.
1
Expression of the results
The sample volume (Vs) is given by the equation:
is plotted against PIPo according to equation (1) . This plot

should yield a straight line usually in the approximate relative
pressure range 0.05 to 0.3. The data are considered
acceptable if the correlation coefficient, r, of the linear
The density (p) is given by the equation: regression is not les s than 0.9975; that is, ¡;l is not les s than
m 0.995 . From the resulting linear plot, the slope, which is
p =- equal to (e - l)I v,J/e, and the intercept, which is equal to
Vs
l/Vme, are evaluated by linear regression analysis. From
The sample conditioning is indicated with the results. these values, v,J/ is calculated as l/(slape + intercept), while e
For example, indicate whether the sample was tested as is or is calculated as (slapelintercept) + l . From the value of Vm so
dried under specific conditions such as those described for determined, the specific surface area, S, in m 2 .g- 1, is
los s on drying. calculated by the equation:
s= VmNa
(2)
m x 22400
M. Specific Surface Area by
Gas Adsorption 1 N
a
Avogadro constant (6.022 x 1023 mol- I ),
effective cross-sectional area of one adsorbate
(Ph. Eur. methad 2. 9.26) molecule, in square metres (0.162 nm2 for
nitrogen and 0.195 nm 2 for lqypton) ,
Introduction
m mass of test powder, in grams,
The specific surface area of a powder is determined by 22400 volume occupied by 1 mole of the adsorbate gas
physical adsorption of a gas on the surface of the solid and at STP allowing for minor departures from the
by calculating the amount of adsorbate gas corresponding to ideal, in millilitres.
a monomolecular layer on the surface . Physical adsorption
results from relatively weak forces (van der Waals forces) A minimum of 3 data points is required. Additional
between the adsorbate gas molecules and the adsorbent measurements may be carried out, especially when non-
surface of the test powder. The determination is usually linearity is obtained at a PIPo value close to 0.3. Because
carried out at the temperature of liquid nitrogen. non-linearity is often obtained at a PIPo value below 0.05,
The amount of gas adsorbed can be measured by a values in this region are not recommended. The test for
volumetric or continuous fiow procedure. linearity, the treatment of the data, and the calculation of the
specific surface area of the sample are described aboye.
Brunauer, Emmett and Teller (BET) theory and Single-point measurement
specific surface area determination
Normally, at least 3 measurements of Va each at different
Multi-point measurement values of PIPo are required for the determination of specific
The data are treated according to the Brunauer, Emmett and surface area by the dynamic fiow gas adsorption technique
Teller (BET) adsorption isotherm equation: (Methad I) or by volumetric gas adsorption (Methad JI).
However, under certain circumstances described below, it
may be acceptable to determine the specific surface area of a
(1)
powder from a single value of Va measured at a single value
ofPlP o such as 0.300 (corresponding to 0.300 mole of
nitro gen or 0.001038 mole fraction of krypton) , using the
P partial vapour pressure of adsorbate gas in following equation for calculating Vn¡:
equilibrium with the surface at 77.4 K (b.p. of liquid
nitrogen), in pascals,
Po saturated pressure of adsorbate gas, in pascals, (3)
Va volume of gas adsorbed at standard temperature and
pressure (STP) [273.15 K and atmospheric pressure The specific surface area is then calculated from the value of
(1.013 x 10 5 Pa)], in millilitres, v'n by equation (2) given aboye.
Vm volume of gas adsorbed at STP to produce an The single-point method may be employed directly for a
apparent monolayer on the sample surface, in series of powder samples of a given material for which the
millilitres,
material constant e is much greater than unity. These
e dimensionless constant that is related to the enthalpy
circumstances may be verified by comparing values of
of adsorption of the adsorbate gas on the powder specific surface area determined by the single-point method
sample.
with that determined by the multiple-point method for the
A value of Va is measured at each of not les s than 3 values of series of powder samp1es. Close similarity between the single-
PIPo' point values and multiple-point values suggests that 11e
approaches zero.
1 This chapeer has undergone pharmacopoeial harnlOnisarion. See chapter The single-point method may be employed indirectly for a
5.8 Pharmacopoeial harmonisation. series of very similar powder samples of a given material for
V-A498 Appendix XVII M 2014
which the material constant e is not infinite but may be AH gases used must be free from moisture.
assumed ro be invariant. Under these circumstances, the QUANTITY OF SAMPLE
error associated with the single-point method can be reduced Accurately weigh a quantity of the test powder such that the
or eliminated by using the multiple-point method ro evaluate total surface of the sample is at least 1 m 2 when the
e for one of the samples of the series from the BET plot, adsorbate is nitrogen and 0.5 m 2 when the adsorbate is
from which e is ca1culated as (1 + slopelintercept). Then V", is krypron.
ca1culated from the single value of Va measured at a single
Lower quantities of sample may be used after appropriate
value of PIPo by the equation: .
validation.
Po
Vm = Va ( p - 1
) [1e + C-
e-1 (
x
P )]
Po (4)
Measurements
Because the amount of gas adsorbed under a given pressure
tends to increase on decreasing the temperature, adsorption
The specific surface area is ca1culated from V,1l by equation measurements are usually made at a low temperature.
(2) given aboye. Measurement is performed at 77.4 K, the boiling point of
liquid nitro gen.
Experimental techniques
METHOD 1: THE DYNAMIC FLOW METHOD
Thissection describes the methods to be used for the sample Principle
preparation, the dynamic ftow gas adsorption technique In the dynamic ftow method (see Figure 2.9.26.-1), the
(Method l) and the volumetric gas adsorption technique recommended adsorbate gas is dry nitrogen or krypron, while
(Method II).
helium is employed as a diluent gas, which is not adsorbed
Sample preparation under the recommended conditions.
OUTGASSING A minimum of 3 mixtures of the appropriate adsorbate gas
Before the specific surface area of the sample can be with helium are required within the PIPo range 0.05 ro 0.30.
determined, it is necessary tó remove gases and vapours that The gas detector-integrator should provide a signal that is
may have become physically adsorbed onto the surface after approximately proportional to the volume of the gas passing
manufacture and during treatment, handling and storage . through it under defined conditions of temperature and
If outgassing is not achieved, the specific surface area may be pressure. For this purpose, a thermal conductivity detector
reduced or may be variable beca use an intermediate area of with an electronic integraror is one among various suitable
the surface is covered with molecules of the previously types. A minirnum of 3 data points within the recommended
adsorbed gases or vapours. The outgassing conditions are range of 0.05 to 0.30 for PIPo is to be determined.
critical for obtaining the required precision and accuracy of
Procedure
specific surface area measurements on pharmaceuticals
A known mixture of the gases, usually nitrogen and helium,
because of the sensitivity of the surface of the materials.
is passed through a thermal conductivity cell, through the
Conditions The outgassing conditions must be sample, again through the thermal conductivity cell and then
demonstrated to yield reproducible BET plots, a constant to a recording potentiometer.
weight of test powder, and no detectable physical or
Irnmerse the sample cell in liquid nitrogen, then the sample
chemical changes in the test powder.
adsorbs nitrogen from the mobile phase. This unbalances the
The outgassing conditions defined by the temperature, thermal conductivity cell, and a pulse is generated on a
pressure and time should be chosen so that the original recorder chart.
surface of the solid is reproduced as closely" as possible.
Remove from the coolant; this gives a desorption peak equal
Outgassing of many substances is often achieved by applying
in area and in the opposite direction to the adsorption peak.
a vacuum, by purging the sample in a ftowing stream of a
Since this is better defined than the adsorption peak, it is the
non-reactive, dry gas, or by applying a desorption-adsorption
one used for the determination.
cycling method. In either case, elevated temperatures are
sometirnes applied ro increase. the rate at which the To effect the calibration, inject a known quantity of
contaminants leave the surface. Caution should be exercised adsorbate into the system, sufficient to give a peak of similar
when outgassing powder samples using elevated temperatures magnitude ro the desorption peak and obtain the· proportion
ro avoid affecting the nature of the surface and the integrity of gas volume per unit peak area.
of the sample. Use a nitrogenlhelium mixture for a single-point
If heating is employed, the recommended temperature and determination and several such mixtures or premixing 2
time of outgassing are as low as possible to achieve streams of gas for a multiple-point deterrnination.
reproducible measurement of specific surface area in an Ca1culation is essentially the same as for the volumetric
acceptable time. For outgassing sensitive samples, other method.
outgassing methods such as the desorption-adsorption cycling METHOD II: THE VOLUMETRIC METHOD
method may be employed. Principie
ADSORBATE In the volumetric method (see Figure 2.9.26.-2), the
The standard technique is the adsorption of nitrogen of recommended adsorbate gas is nitrogen which is admitted
analytical quality at liquid nitrogen temperature . into the evacuated space aboye the previously outgassed
For powders of low specific surface area « 0.2 m 2.g- 1) the powder sample to give a defined equilibrium pressure, P, of
proportion adsorbed is low. In such cases the use of krypron the gas. The use of a diluent gas, such as helium, is therefore
at liquid nitrogen temperature is preferred because the low unnecessary, although helium may be employed for other
vapour pressure exerted by this gas greatly reduces error. purposes, such as to measure the dead volume.
The use of larger sample quantities where feasible (equivalent Since only pure adsorbate gas, instead of a gas mixture, is
to 1 m 2 or greater total surface area using nitrogen) may employed, interfering effects of thermal diffusion are avoided
compensate for the errors in determining low surface areas. in this method.
2014 Appendix XVII M V-A499
Sel! seals quick connection
o ring seals
Calibrating
Septum
Path
Flow
meter ,
I ~
selection Diffusion
Flow
1 valva baffle
control
valve
Outgassing
Sample
station
cell
Differential
flow
Short Long
controller
path path
ballast ballast
On-off
valve Digital +-
A display B
Vent
Gas inlet
Detector Detector
Figure 2.9.26.-1. - Schematic diagram ofthe dynamic flow method apparatus
9
To cold traps and
4 3 vacuum pumps
7 10 11
8
Helium
reservoir
......
......
Vacuum
Vapour
J~:::Qtc=~ Air
pressure
manometer
Figure 2.9.26.-2. - Schematic diagram of the volumetric method apparatus
Procedure is, by means of reference and sample tubes connected by a

Admit a small amount of dry nittogen into the sample tube differential transducer. The adsorption of nittogen gas is then
to prevent contamination of the clean surface, remove the measured as described below.
sample tube, insert the stopper, and weigh it. Calculate the Raise a Dewar vessel containing liquid nitro gen at 77.4 K up
weight of the sample. Attach the sample tube to the to a defined point on the sample cel!. Admit a sufficient
volumetric apparatus. Cautiously evacuate the sample down volume of adsorbate gas to give the lowest desired relative
to the specified pressure (e.g. between 2 Pa and 10 Pa). pressure. Measure the volume adsorbed, Va. For multipoint
Altematively, sorne insttuments operate by evacuating to a measurements, repeat the measurement of Va at successively
defined rate ofpressure change (e.g. less than 13 Pa/30 s) higher PIPo values. When nitrogen is used as the adsorbate
and holding for a defined period of time before commencing gas, PIPo values of 0.10, 0.20, and 0.30 are often suitable.
the next step .
If the principIe of operation of the instrument requires the Reference materials
determination of the dead volume in the sample tube, for Periodically verify the functioning of the apparatus using
example, by the admission of a non-adsorbed gas, such as appropriate reference material s of known surface area, such
he1ium, this procedure is carried out at this point, followed as cx-alumina, which should have a specific surface area
by evacuation of the sample. The determination of dead similar ro that of the sample ro be examined.
volume may be avoided using difference measurements, that
V-ASOO Appendix XVII N 2014
N. Powder Flow 1 experimental variables:

- the height of the 'funnel' through which the powder
(Ph. Eur. method 2.9.36) passes may be fixed relative to the base, or the height may
The widespread use of powders in the pharmaceutical be varied as the piJe forms;
industry has generated a variety of methods for characterising - the base upon which the piJe forms may be of fixed
powder flow. Not surprisingly, scores of references appear in diameter or the diameter of the powder cone may be
the pharmaceutical Jiterature, attempting to corre late the aJIowed to vary as the pile forms .
various measures of powder flow to manufacturing
properties. The development of such a variety of test Variations in angle of repose methods
methods was inevitable; powder behavior is multifaceted and Variations of the aboye methods have also been used to sorne
thus complicates the effort to characterise powder flow. extent in the pharmaceutical literature:
The purpose of this chapter is to review the methods for - drained angle 01 repose: this is determined by aJIowing an
characterising powder flow that have appeared most excess quantity of material positioned aboye a fixed
frequently in the pharmaceutical Jiterature. In addition, while diameter base to "drain" from the container. Formation
it is clear that no single and simple test method can of a con e of powder on the fixed diameter base allows
adequately characterise the flow properties of pharmaceutical determination of the drained angle of repose;
powders, this chapter proposes the standardisation of test - dynamic angle 01 repose: this is determined by filling a
methods that may be valuable during pharmaceutical cyJinder (with a clear, flat cover on one end) and rotating
development. it at a specified speed. The dynamic angle of repose is the
4 commonly reported methods for testing powder flow are: angle (relative to the horizontal) formed by the flowing
- angle of repose, powder. The intemal angle of kinetic friction is defined by
the plane separating those particJes sliding down the top
- compressibiJity index or Hausner ratio,
layer of the powder and those particles that are rotating
- flow rate through an orifice, with the drum (with roughened surface) .
- shear cell.
General scale offlowability for angle of repose
In addition, numerous variations of each of these basic
While there is sorne variation in the quaJitative description of
methods are avaiJable. Given the number of test methods
powder flow using the angle of repose, much of the
and variations, standardising the test methodology, where
pharmaceutical Jiterature appears to be consistent with the
possible, would be advantageous.
classification by Carr2 , which is shown in Table 2.9.36.-l.
With this goal in mind, the most frequently used methods There are examples in the literature of formulations with an
are discussed below. Important experimental considerations angle of repose in the range of 40-50 degrees that
are identified and recommendations are made regarding manufactured satisfactorily. When the angle of repose
standardisation of the methods. In general, any method of exceeds 50 degrees, the flow is rarely acceptable for
measuring powder flow must be practical, useful, manufacturing purposes.
reproducible and sensitive, and must yield meaningful results.
It bears repeating that no simple powder flow method will Table 2.9.36.-1. - Flow properties and corresponding angles of
adequately or completely characterise the wide range of flow repose(2 )
properties experienced in the pharmaceutical industry.
Flow property Angle oC repose (degrees)
An appropriate strategy may well be the use of multiple
standardised test methods to characterise the various aspects Excellent 25-30
of powder f10w as needed by the pharmaceutical scientist. Good 31-35
Angle of Repose Fair (aid not needed) 36-40
The angle of repose has been used in several branches of Passable (may hang up) 41-45
science to characterise the flow properties of soJids. Angle of POOl' (must agita te. vibra te) 46·55
repose is a characteristic related to interparticulate friction, or
Very pOOl' 56·65
resistance to movement between particles. Angle of repose
test results are reported to be very dependent upon the Very. very pOOl' > 66
method used. Experimental difficulties arise due to

segregation of material and consolidation or aeration of the
Experimental considerations for angle of repose
powder as the cone is formed. Despite its difficulties, the
method continues to be used in the pharmaceutical industry, Angle of repose is not an intrinsic property of the powder,
and a number of examples demonstrating its value in that is to say, it is very much dependent upon the method
predicting manufacturing problems appear in the Jiterature. used to form the cone of powder. On this subject, the
existing literature raises these important considerations:
The angle of repose is the constant, three-dimensional angle
(relative to the horizontal base) assumed by a cone-Jike piJe - the peak of the cone of powder can be distorted by the
of material formed by any of several different methods, impact of powder from aboye. By carefully building the
described briefly below. powder cone, the distortion caused by impact can be
mjnimised;
Basic methods for angle of repose
- the nature of the base upon which the powder cone is
A variety of angle of repose test methods are described in the formed influences the angle of repose. Ir is recommended
Jiterature. The most common methods for determining the that the powder cone be formed on a 'common base',
sta tic angle of repose can be classified based on 2 important which can be achieved by forming the con e of powder on
a layer of powder. This can be done by using a base of
I This chapter has undergone phannacopoeial hannonisarion. See chapter
5.8. Pannacopoeial hannonisation. 2 Carr RL: Evalualing jlow propenies of solids. Chem. Eng 1965,- 72 163
2014 Appendix XVII N V-AS01
fixed diameter with a protruding outer edge to retain a Table 2.9.36.-2. - Scale offlowabilitylZJ
layer of powder upon which the cone is formed. Compressibility index Flow character Hansner ratio
(per cent)
Recommended procedure for angle of repose
}.lO Excellent 1.00·1.11
Form the angle of repose on a fixed base with a retaining lip
11-15 Good 1.12-1.18
to reta in a layer of powder on the base. The base must be
free of vibration. Vary the height of the funnel to carefuIly 16-20 Fair 1.19-1.25
build up a symmetrical cone of powder. Care must be taken 21-25 Passab!e 1.26-1.34
to prevent vibration as the funnel is moved. The funnel
26-31 Poor 1.35·1.45
height is maintained at approximately 2-4 cm from the top of
the powder pile as it is being formed in order to minimise the 32-37 Very poor 1.46-1.59
impact of faIling powder on the tip of the cone. If a > 38 Very, very poor > 1.60
symmetrical con e of powder cannot be successfuIly or
reproducibly prepared, this method is not appropriate.
Determine the angle of repose by measuring the height of the Experimental considerations for the compressibility
cone of powder and caIculating the angle of repose, 0., from index and Hausner ratio
the foIlowing equation:
Compressibility index and Hausner ratio are not intrinsic
properties of the powder, that is to say, they are dependent
height upon the methodology used. The existing literature points
tan (a) = 05 b
. X ase out several important considerations affecting the
determination of me unsettled apparent volume, Va, of the
final tapped volume, V¡, of the bulk density, Pbulk, and of the
Compressibility index and Hausner ratio
tapped density, Prapped:
In recent years the compressibility index and the cIosely - the diameter of the cylinder used,
reIated Hausner ratio have become the simple, fast, and
popular methods of predicting powder flow characteristics. - the number of times the powder is tapped to achieve the
The compressibility index has been proposed as an indirect tapped density,
measure of bulk density, size and shape, surface area, - the mass of material used in the test,
moisture content, and cohesiveness of materials, because aIl - rotation of the sample during tapping.
of these can influence the observed compressibility indexo Recommended procedure for compressibility index
The compressibility index and the Hausner ratio are and Hausner ratio
determined by measuring both the buIk volume and tapped
volume of a powder. Use a 250 mL volumetric cylinder with a test sample mass of
100 g. SmaIler amounts and volumes may be used, but
Basic methods for compressibility index and Hausner variations in the method must be described with the results.
ratio An average of 3 determinations is recommended.
While there are sorne variations in the method of determining
the compressibility index and Hausner ratio, the basic Flow through an orifice
procedure is to measure the unsettled apparent volume, (Vo), The flow rate of a material depends upon many factors, sorne
and the final tapped volume, (V¡), of the powder after of which are particIe-related and sorne related to the process.
tapping the material until no further volume changes occur. Monitoring the rate of flow of material through an orifice has
The compressibility index and the Hausner ratio are been proposed as a better measure of powder flowability.
caIculated as foIlows: Of particular significance is the utility of monitoring flow
continuously, since puIsating flow patterns have been
Va - VI observed even for free-flowing materials. Changes in ftow rate
Compressibility Index := 100 X - - = ---'-
as the container empties can also be observed. Empírical
Va
equations reIating flow rate to the diameter of the opening,
· Va
H ausner R atw = VI particle size, and particle density have been determined.
However, determining the ftow rate through an orifice is
useful only with free-ftowing materíals.
AItematively, the compressibility index and Hausner ratio The flow rate through an orífice is generaIly measured as the
may be caIculated using measured values of bulk density mas s per time flowing from any of a number of types of
(Pbulk) and tapped density (Pzappec¡) as foIlows: containers (cylinders, funnels, hoppers). Measurement of the
flow rate can be in discrete increments or continuous.
Compressibility Index = 100 X ptapped - pbulk Basic methods for flow through an orifice
ptapped
There are a variety of methods described in the literature.
H ausner Ratio = Ptapped The most common for determining the ftow rate through an
Pbulk orífice can be classified based on 3 important experimental
variables:
In a variation of these methods, the rate of consolidation is - the type of container used to contain the powder.
sometimes measured rather than, or in addition to, the Common containers are cylinders, funnels, and hoppers
change in volume that occurs on tapping. For the from production equipment;
compressibility index and the Hausner ratio, the generaIly - the size and shape of the orifice used. The orífice diameter
accepted scale offtowability is given in Table 2.9.36.-2. and shape are crítical factors in determining powder flow
rate;
V-A502 Appendix XVII N 2014
- the method of measuring powder fiow rateoFlow rate can pattern. Rate measurement can be either discrete or
be measured continuously using an electronic balance continuous. Continuous measurement using an electronic
with sorne sort of recording device (strip chart recorder, balance can more effectively detect momentary fiow rate
computer). It can also be measured in discrete samples variations.
(for example, the time it takes for 100 g of powder to pass
through the orifice to the nearest tenth of a second or the
Shear cell methods
amount of powder passing through the orifice in lOs to In an effort to put powder fiow studies and hopper design on
the nearest tenth of a gram). a more fundamental basis, a variety of powder shear testers
and methods that permit more thorough and precisely
Variations in methodslor flow through an orifice defined assessment of powder fiow properties have been
Either mass fiow rate or volume fiow rate can be determined. developed. Shear cell methodology has been used extensively
Mass fiow rate is the easier of the methods, but it biases the in the study of pharmaceutical materials. From these
results in favour of high-density materials. Since die fill is methods, a wide variety of parameters can be obtained,
volumetric, determining volume fiow rate may be preferable. ineluding the yield loci representing the shear stress-shear
A vibrator is occasionally attached to facilitate fiow from the strain relationship, the angle of intemal friction, the
container, however, this appears to complicate interpretation unconfined yield strength, the tensile strength, and a variety
of results. A moving orifice device has been proposed to of derived parameters such as the fiow factor and other
more elosely simulate rotary press conditions. The minimum fiowability indices. Because of the ability to control
diameter orifice through which powder fiows can also be experimental parameters more precisely, fiow properties can
identified. also be determined as a function of consolidation load, time,
General scale 01 flowability lor flow through an orifice and other environmental conditions. These methods have
N o general scale is available because fiow rate is critically been successfully used to determine critical hopper and bin
dependent on the method used to measure it. Comparison parameters.
between published results is difficult. Basic methods lor shear cell
Experimental considerations lor flow through an One type of shear cell is the cylindrical shear cell which is
orifice split horizontally, forming a shear plane between the lower
Flow rate through an orifice is not an intrinsic property of stationary base and the upper moveable portion of the shear
the powder. It is very much dependent upon the cell ringo After powder bed consolidation in the shear cell
methodology used. The existing literature points out several (using a well-defined procedure), the force necessary to shear
important considerations affecting these methods: the powder bed by moving the upper ring is determined.
Annular shear cell designs offer sorne advantages over the
- the diameter and shape of the orifice,
cylindrical shear cell design, ineluding the need for less
- the type of container material (metal, glass, plastic), material. A disadvantage, however, is that because of its
- the diameter and height of the powder bed. design, the powder hed is not sheared as uniformly because
Recommended procedure lor flow through an orifice material on the outside of the annulus is sheared more than
material in the inner regíon. A third type of shear cell (plate-
Flow rate through an orifice can be used onJy for materials
type) consists of a thin sandwich of powder between a lower
that have sorne capacity to fiow. It is not useful for cohesive
stationary rough surface and an upper rough surface that is
materials. Provided that the height of the powder bed (the
moveable.
'head' of powder) is much greater than the diameter of the
orifice, the fiow rate is virtually independent of the powder All of the shear cell methods have their advantages and
head. It is advisable to use a cylinder as the container, disadvantages, but a detailed review is beyond the scope of
because the walls of the container must have little effect on this chapter. As with the other methods for characterising
fiow. This configuration results in fiow rate being determined powder fiow, many variations are described in the literature.
by the movement of powder over powder, rather than . A significant advantage of shear cell methodology in general
powder along the wall of the container. Powder fiow rate is a greater degree of experimental control. The methodology
often increases when the height of the powder colurnn is less generally is rather time-consuming and requires significant
than twice the diameter of the column. The orifice must be amoun~s of material and a well-trained operator.
circular and the cylinder must be free of vibration. General Recommendations lor shear cell
guidelines for dime~sions of the cylinder are as follows: The many existing shear cell configurations and test methods
- di ame ter of the opening greater than 6 times the diameter provide a wealth of data and can be used very effectively to
of the partieles, characterise powder fiow. They are also helpful in the design
- diameter of the cylinder greater than twice the diameter of of equipment such as hoppers and bins. Because of the
the opening. diversity of available equipment and experimental procedures,
Use of a hopper as the container may be appropriate and no specific recommendations regarding methodology are
representative of fiow in a production situation. It is not presented in this chapter. It is recommended that the results
advisable to use a funnel, particularly one with a stem, of powder fiow characterisation using shear cell methodology
because fiow rate will be determined by the size and length inelude a complete description of equipment and
of the stem as well as the friction between the stem and the methodology used.
powder. A truncated cone may be appropriate, but fiow will
be infiuenced by the powder-wall friction coefficient, thus,
selection of an appropriate construction material is
important.
For the opening in the cylinder, use a fiat-faced bottom plate
with the option to vary orifice diameter to provide maximurn
f1exibility and better ensure a powder-over-powder fiow
2014 Appendix XVII O V-A503
O. Optical Microscopy1 of magnification. The apparent size of a photographic image

is infiuenced by the exposure, development, and printing
(Ph. Eur. methad 2.9.3 7) processes as well as by the resolving power of the
Optical microscopy for particle characterisation can generally microscope.
be applied ro particles of 1 ~m and greater. The lower Iimit Preparation of the mount The mounting medium will
is imposed by the resolving power of the microscope. vary according ro the physical properties of the test sample.
The upper Iimit is less definite and is determined by the Sufficient, but not excessive, contrast between the sample
increased difficulty associated with the characterisation of and the mounting medium is required ro ensure adequate
larger particles. Various alternative techniques are available detail of the sample edge. The particles must rest in one
for particle characterisation outside the applicable range of plane and be adequately dispersed ro distinguish individual
optical microscopy. Optical microscopy is particularly useful particles of interest. Furthermore, the particles must be
for characterising particles that are not spherical. This representative of the distribution of sizes in the material and
method may also serve as a base for the calibration of faster must not be altered during preparation of the mount. Care
and more routine methods that may be developed. must be taken ro ensure that this important requirement is
Apparatus Use a microscope that is stable and protected met. Selection of the mounting medium must include a
from vibration. The microscope magnification Cproduct of consideration of the analyte solubility.
the objective magnification, ocular magnification, and Crystallinity characterisation The crystallinity of a
additional magnifying components) must be sufficient to material may be characterised to determine compliance with
allow adequate characterisation of the smallest particles to be the crystallinity requirement where stated in the individual
c1assified in the test sample. The greatest numerical aperture monograph of a drug substance. Unless otherwise specified
of the objective is sought for each magnification range. in the individual monograph, mount a few particles of the
Polarising filters may be used in conjunction with suitable sample in mineral oi! on a c1ean glass slide. Examine the
analysers and retardation plates. Colour filters of relatively mixture using a polarising microscope: the particles show
narrow spectral transmission are used with achromatic birefringence Cinterference colors) and extinction positions
objectives, and are preferable with apochromats; they are when the microscope stage is revolved.
required for appropriate colour rendition in
Lirnit test ofparticle size by microscopy Weigh a
photomicrography. Condensers, corrected at least for
suitable quantity of the powder to be examined Cfor example,
spherical aberration are used in the microscope substage and
10-100 mg), and suspend it in 10 mL of a suitable medium
with the lamp . The numerical aperture of the substage
in which the powder do es not dissolve, adding, if necessary,
condenser matches that of the objective under the conditions
a wetting agent. A homogeneous suspension of particles can
of use; this is affected by the actual aperture of the
be maintained by suspending the particles in a medium of
condenser diaphragm and the presence of immersion oils.
similar or matching density and by providing adequate
Adjustment The precise alignment of all elements of the agitation. Introduce a portion of the homogeneous
optical system and proper focusing are essential. suspension into a suitable counting cell, and scan under a
The focusing of the elements is done in accordance with the microscope an area corresponding ro not les s than 1O ~g of
recommendations of the microscope manufacturero Critical the powder robe examined. Count all the particles having a
axial alignment is recommended. maximum dimension greater than the prescribed size Iimit.
Illumination A requirement for good iIIumination is a The size Iimit and the permitted number of particles
uniform and adjustable intensity of light over the entire field exceeding the limit are defined for each substance.
of view; K6hler illumination is preferred. With coloured Particle size characterisation The measurement of
particles, choose the colour of the filters so as ro control the particle size varies in complexity depending on the shape of
contrast and detail of the image. the particle, and the number of particles characterised must
Visual characterisation The magnification and numerical be sufficient ro ensure an acceptable level of uncertainty in
aperrure must be sufficiently high ro allow adequate the measured parameters. Additional information on particle
resolution of the images of the particles to be characterised.
Determine the actual magnification using a calibrated stage
micrometer to calibrate an ocular micrometer. Errors can be
minimised if the magnification is suffici~nt that the image of
the particle is at least 10 ocular divisions . Each objective
must be calibrated separately. To calibrate the ocular scale,
the stage micrometer scale and the ocular scale must be ____--------l~ Feret's diameter
aligned. In this way, a precise determination of the distance
between ocular stage divisions can be made. Several different
magnifications may be necessary to characterise materials
having a wide particle size distribution.
Photographic characterisation If particle size is ro be Martin's diameter
determined by photographic methods, take care to ensure
that the object is sharply focused at the plane of the
phorographic emulsion. Determine the actual magnification
by photographing a calibrated stage micrometer, using
photographic film of sufficient speed, resolving power, and
contrasto Exposure and processing must be identical for
phorographs of both the test sample and the determination
1 This chapter has undergone pharmacopoeial harmonisation. See chapter Figure 2.9.37.-1. - Commonly used measurements of particle
5.8. Pharmacopoeial harmonisation. size
V-A504 Appendix XVII P 2014
Flake
Equant
Plate
Columnar ~
I~------ Lath
Figure 2.9.37.-2. - Commonly used descriptions of particle shape
size measurement, sample size, and data analysis is available, - lamellar. stacked plates,
for example, in ISO 9276. For spherical particles, size is - aggregate: mas s of adhered particles,
defined by the diameter. For irregular particles, a variety of - agglomerate: fused or cemented particles,
definitions of particle size existo In general, for irregularly
- conglomerate: mixture of 2 or more types of particles,
shaped particles, characterisation of particle size must also
include inforrnation on the type of diameter measured as - spherulite: radial cluster,
well as inforrnation on particle shape. Several commonly - drusy: particle covered with tiny particles.
used measurements of particle size are defined in Figure Particle condition may be described by the following terrns:
2.9.37.-1. - edges: angular, rounded, smooth, sharp, fractured,
- Feret's diameter: the distance between imaginary parallel - optical: color (using proper color balancing filters),
lines tangent to a randomly oriented particle and transparent, translucent, opaque,
perpendicular to the ocular scale,
- defects: occlusions, inclusions.
- Martin's diameter. the diameter of the particle at the point
that divides a randomly oriented particle into 2 equal Surface characteristics may be described as:
projected are as, - cracked: partial split, break, or fissure,
- projected area diameter. the diameter of a circle that has the - smooth: free of irregularities, roughness, or projections,
same projected area as the particle, - porous: having openings or passageways,
- length: the longest dimension from edge to edge of a - rough: bumpy, uneven, not smooth,
particle oriented parallel to the ocular sea le, . - pitted: small indentations.
- width: the longest dimension of the particle measured at
right angles lO the length.
Particle shape characterisation For irregularly shaped
particles, characterisation of particle size must also include P. Particle Size Analysis by Laser Light
inforrnation on particle shape. The homogeneity of the
powder must be checked using appropriate magnification.
Diffraction
The following defines sorne commonly used descriptors of (Ph. Eur. method 2.9.31)
particle shape (see Figure 2.9.37.-2). The method is based on the ISO standards 13320-1 (1999)
- acicular. slender, needle-like particle of similar width and and 9276-1(1998) .
thickness,
Introduction
- columnar. long, thin particle with a width and thickness
that are greater than those of an acicular particle, The laser Iight diffraction technique used for the
deterrnination of particle-size distribution is based on the
- flake: thin, fiat particle of similar length and width,
analysis of the diffraction pattem produced when particles are
- plate: fiat particle of similar length and width but with exposed to a beam of monochromatic Iight. HislOrically, the
greater thickness than a fiake particle, early laser diffraction instruments only used scattering at
- lath: long, thin, blade-like particle, small angles. However, the technique has since been
- equant: particle of similar length, width, and thickness; broadened to include laser light scattering in a wider angular
both cubical and spherical particles are included. range and application of the Mie theory, in addition lO the
General observations A particle is generally considered to Fraunhofer approximation and anomalous diffraction.
be the smallest discrete unit. A particle may be a liquid or The technique cannot distinguish between scattering by
semi-solid droplet; a single crystal or polycrystalline; single particles and scattering by c1usters of primary particles,
amorphous or an agglomerate. Particles may be associated. i.e. by agglomerates or aggregates . As most particulate
This degree of association may be described by the following samples contain agglomerates or aggregates and as the focus
terrns: of interest is generally on the size distribution of primary
2014 Appendix XVII P V-ASOS
9 11
10
7 8
(1) 1
3
I
2
4
5
1. Obscuration detector 5. Scattered light not collected by lens (4) 9. Working distance of lens (4)
2. Scattered beam 6. Partic1e ensemble 10. Multi-element detector
3. Direct beam 7. Light source laser 11. Focal distance of lens (4)
4. Fourier lens 8. Beam processing unit
Figure 2.9.31.-1. - Example of a set-up of a laser light diffraction instrument
particles, the cIusters are usually dispersed into primary the collecting lens and thus, in a converging beam.
particles before measurement. The advantage of the conventional set-up is that a reasonable
For non-spherical particles, an equivalent sphere-size path length for the sample is allowed within the working
distribution is obtained because the technique assumes distance of the lens. The second set-up allows onIy small
spherical particIes in its optical model. The resulting particIe- path lengths but enables measurement of scattered light at
size distribution may differ from those obtained by methods larger angles, which is useful when submicron particles are
based on other physical principIes (e.g. sedimentation, present.
sieving) . The interaction of the incident light beam and the ensemble
This chapter provides guidance for the measurement of size of dispersed particles results in a scattering pattem with
distributions of particles in different dispersed systems, for different light intensities at various angles. The total angular
example, powders, sprays, aerosols, suspensions, emulsions, intensity distribution, consisting of both direct and scattered
and gas bubbles in liquids, through analysis of their angular light, is then focused onto a multi-element detector by a lens
light-scattering pattems. It do es not address specific or a series of lenses. These lenses create a scattering pattem
requirements of particle size measurement of specific that, within limits, does not depend on the location of the
products. particles in the light beam. Hence, the continuous angular
intensity distribution is converted into a discrete spatial
PrincipIe intensity distribution on a set of detector elements.
A representative sample, dispersed at an adequate It is assumed that the measured scattering pattem of the
concentration in a suitable liquid or gas, is passed through a particIe ensemble is identical to the sum of the pattems from
beam of monochromatic light, usually a laser. The light all individual single scattering particIes presented in random
scattered by the particles at various angles is measured by a relative positions. Note that only a limited angular range of
multi-element detector. Numerical values representing the scattered light is collected by the lens(es) and, therefore, by
scattering pattem are then recorded for subsequent analysis. the detector.
These scattering pattem values are then transformed, using
an appropriate optical model and mathematical procedure, to Development of the method
yield the proportion of total·volume to a discrete number of The measurement of particIe size by laser diffraction can give
size classes, forming a volumetric particle-size distribution. reproducible data, eVen in the sub-micron region, provided
the instrument used and the sample tested are carefully
Instrument controlled to limit variability of the test conditions
The instrument is located in an environment where it is not (e.g. dispersion medium, method of preparation of the
affected by electrical noise, mechanical vibrations, sample dispersion) .
temperature fiuctuations, humidity or direct bright light. Traditionally, the measurement of particle size using laser
An example of a set-up of a laser light diffraction instrument diffraction has been limited to particIes in the range of
is given in Figure 2.9.31.-1. Other equipment may be used. approximately 0.1 flm to 3 mm. Because of recent advances
The instrument comprises a laser light source, beam in lens and equipment design, newer instruments are capable
processing optics, a sample measurement region (or cel!), a of exceeding this range routinely. With the validation report
Fourier lens, and a multi-element detector for measuring the the user demonstrates the applicability of the method for its
scattered light pattem. A data system is also required for intended use.
deconvolution of the scattering data into a volumetric size Sampling The sampling technique must be adequate to
distribution and associated data analysis and reporting. obtain a representative sample of a suitable volume for the
The particles can enter the laser beam in 2 positions. In the particle-size measurement. Sample splitting techniques such
conventional case the particIes enter the parallel beam before as rotating riffier or the cone and quartering method may be
the collecting lens and within its working distance. applied .
In so-called reversed Fourier optics the particles enter behind
V A506 Appendix XVII P 2014
Evaluation of the dispersion procedure Inspect the - be compatible with the materials used in the instrument
sample ro be analysed, visually or with the aid of a (such as O-rings, gaskets, tubing, etc.);
microscope, to estimate its size range and partic1e shape. - possess a suitable viscosity to facilitate recirculation,
The dispersion procedure must be adjusted to the purpose of stirring and filtration.
the measurement. The purpose may be such that it is Surfactants andJor dispersing aids are often used to wet the
preferable to deagglomerate c1usters into primary partic1es as partic1es and to stabilise the dispersion. For weak acids and
far as possible, or it may be desirable to retain c1usters as weak bases, buffering of the dispersing medium at low or
intact as possible. In this sense, the partic1es of interest may high pH respectively can assist in identifying a suitable
be either primary partic1es or c1usters. dispersant.
For the development of a method it is highly advisable to A preliminary check of the dispersion quality can be
check that comminution of the partic1es does not occur, and performed by visual or microscopic inspection. It is also
conversely, that dispersion of partic1es or c1usters is possible to take fractional samples out of a well-mixed stock
satisfactory. This can usually be done by changing the dispersion. Such stock dispersions are formed by adding a
dispersing energy and monitoring the change of the partic1e- liquid to the sample while mixing it with, for example, a glass
size distribution. The measured size distribution must not rod, a spatula or a vortex mixer. Care must be taken to
change significantly when the sample is well dispersed and ensure the transfer of a representative sample and that
the partic1es are neither fragile nor soluble. Moreover, if the settling of larger partic1es does not occur. Therefore a sample
manufacturing process (e.g. crystallisation, milling) of the paste is prepared or sampling is carried out quickly from a
material has changed, the applicability of the method must suspension maintained under agitation.
be verified (e.g. by microscopic comparison).
Optimisation of the gas dispersion For sprays and dry
Sprays, aerosols and gas bubbles in a liquid should be powder dispersions, a compres sed gas free from oil, water
measured directly, provided that their concentration is and partic1es may be used. To remove such material s from
adequate, beca use sampling or dilution generally alters the the compressed gas, a dryer with a filter can be used.
partic1e-size distribution. Any vacuum unit should be located away from the
In other cases (such as emulsions, pastes and powders), measurement zone, so that its output does not disturb the
representative samples may be dispersed in suitable liquids. measurement.
Dispersing aids (wetting agents, stabilisers) andJor Determination of the concentration range In order to
mecharucal force s (e.g. agitation, sonication) are often produce an acceptable signal-to-noise ratio in the detector,
applied for deagglomeration or deaggregation of c1usters and the partic1e concentration in the dispersion must exceed a
stabilisation of the dispersion. For these liquid dispersions, a minimum leve!. Likewise, it must be below a maximum level
recirculating system is most commonly used, consisting of an in order to avoid multiple scattering. The concentration
optical measuring cell, a dispersion bath usually equipped range is infiuenced by the width of the laser beam, the path
with stirrer and ultrasonic elements, a pump, and tubing. length of the measurement zone, the optical properties of the
Non-recirculating, stirred cells are useful when only small partic1es, and the sensitivity of the detector elements.
amounts of a sample are available or when special dispersion
In view of the aboye, measurements must be performed at
liquids are used.
different partic1e concentrations to determine the appropriate
Dry powders can also be converted into aerosols through the concentration range for any typical sample of material. (Note:
use of suitable dry powder dispersers, which apply in different instruments, partic1e concentrations are usually
mechanical force for deagglomeration or deaggregation. represented by differently scaled and differently named
Generally, the dispersers use the energy of compressed gas or numbers, e.g. obscuration, optical concentration,
. the differential pressure of a vacuum to disperse the partic1es proportional number of total mass).
to an aerosol, which is blown through the measuring zone,
Determination of the measuring time The time of
usually into the inlet of a vacuum unit that collects the
measurement, the reading time of the detector and the
partic1es. However, for free fiowing, coarser partic1es or
acquisition frequency are determined experimentally in
granules the effect of gravity may be sufficient to disperse the
accordance with the required precision. Generally, the time
partic1es adequately.
for measurement permits a large number of detector scans or
If the maximum partic1e size of the sample exceeds the sweeps at short time intervals.
measuring range of the instrument, the material that is too
Selection of an appropriate optical model Most
coarse can be removed by sieving and the mas s and
instruments use either the Fraunhofer or the Mie theory,
percentage of removed material are reported. However, after
thougb other approximation theories are sometimes applied
pre-sieving, note that the sample is no longer representative,
for calculation of the scattering matrix. The choice of the
unless otherwise proven.
theoretical roo del depends on the intended application and
Optimisation of the liquid dispersion Liquids, the different assumptions (size, absorbance, refractive index,
surfactants, and dispersing aids used to disperse powders roughness, crystal orientation, mixture, etc.) made for the
must: test material. If the refractive index values (real and
- be transparent at the laser wavelength and practically free imaginary parts for the used wavelength) are not exactly
from air bubbles or partic1es; known, then the Fraunhofer approximation or the Mie
- have a refra<;:tive index that differs from that of the test theory with a realistic estimate of the refractive index can be
material; used. The former has the advantages that it is simple and it
- be non-solvent of the test material (pure liquid or pre- does not need refractive index values; the latter usually
filtered, saturated solution); provides less-biased partic1e-size distributions for small
partic1es . For instance, if the Fraunhofer model is used for
- not alter the size of the test materials (e.g. by solubility,
samples containing an appreciable amount of small,
solubility enhancement, or recrystallisation effects);
transparent partic1es, a significantly larger amount of small
- favour easy formation and stability of the dispersion; partic1es may be calculated. In order to obtain traceable
2014 Appendix XVII P V-AS07
results, it is essential to document the refractive index values dispersions; such effects can cause erroneous partic1e-size
used, since small differences in the values assumed for the distributions.
real and imaginary part of the complex refractive index may Measurement of the light scattering of dispersed
cause significant differences in the resulting particle-size sample(s) After proper alignment of the optical part of the
distributions. Small values of the imaginary part of the instrument, a blank measurement of the partic1e-free
refractive index (about 0.01-0.1 i) are often applied to allow dispersion medium must be performed using the same
the correction of the absorbance for the surface roughness of method as that used for the measurement of the sample.
the particles. It should be noted, in general, that the optical The background signal must be below an appropriate
properties of the substance to be tested, as well as the threshold. The detector data are saved in order to substract
structure (e.g. shape, surface roughness and porosity), bear them later from the data obtained with the sample.
upon the final resulto The sample dispersion is measured according to the
Validation Typically, the validity of a procedure may be developed method.
assessed by the evaluation of its specificity, linearity, range, For each detector element, an average signal is calculated,
accuracy, precision and robustness. In particle-size analysis sometimes together with Íts standard deviation.
by laser light diffraction, specificity as defined by ICH is not The magnitude of the signal from each detector element
applicable as it is not possible to discriminate between depends upon the detection area, the light intensity and the
different components in a sample, nor is it possible to quantum efficiency. The co-ordinates (size and position) of
discriminate agglomerates from dispersed particles unless the detector elements together with the focal distance of the
properly complemented by microscopic techniques. lens determine the range of scartering angles for each
Exploring a linear relationship between concentration and element. Most instruments also measure the intensity of the
response, or a mathematical model for interpolation, is not central (unscattered) laser beam. The ratio of the intensity of
applicable to this procedure. Rather than evaluating linearity, a dispersed sample to that in its absence (a blank
this method requires the definition of a concentration range measurement) indica tes the proportion of scattered light and
within which the result of the measurements do es not vary hence the partic1e concentration.
significantly. Concentrations below that range produce an Conversion of scattering pattern into particle-size
error due to a poor signal-to-noise ratio, while concentrations distrihution This deconvolution step is the inverse of the
above that range produce an error due to multiple scartering. calculation of a scattering pattem for a given partic1e-size
The range depends mostly on the instrument hardware. distribution. The assumption of spherical partic1e shape is
Accuracy should be confirmed through an appropriate particularly important as most algorithms use the
instrument qualification and comparison with microscopy, mathematical solution for scartering from spherical partic1es.
while precision may be assessed by means of a repeatability Furthermore, the measured data always contain sorne
determination. random and systematic errors, which may vitiate the size
The artainable repeatability of the method mainly depends distributions. Several mathematical procedures have been
on the characteristics of the material (milledlnot milled, developed for use in the available instruments. They contain
robustlfragile, width of its size distribution, etc.), whereas the sorne weighting of deviations between measured and
required repeatability depends on the purpose of the calculated scattering parterns (e.g. least squares), sorne
measurement. Mandatory limits cannot be specified in this constraints (e.g. non-negativity for amounts of partic1es),
chapter, as repeatabilities (different sample preparations) may and/or sorne smoothing of the size distribution curve.
vary appreciably from one substance to another. However, it The algorithms used are specific to each make and model of
is good practice to aim at acceptance criteria for repeatability equipment, and are proprietary. The differences in the
such as Srd ::; 10 per cent [17 = 6J for any central value of the algorithms between different instruments may give rise to
distribution (e.g. for xso). Values at the sides of the differences in the calculated partic1e-size distributions.
distribution (e.g. x¡O and X90) are oriented towards less
Replicates The number of replicate measurements (with
stringent acceptance criteria such as Srel ::; 15 per cent
individual sample preparations) to be performed depends on
[17 = 6J. Below 10 ~lm, these values must be doubled.
the required measurement precision. It is recommended to
Robustness may be tested during the selection and
set this number in a substance-specific method.
optimisation of the dispersion media and forces . The change
of the dispersing energy may be monitored by the change in
Reporting of results
the particle-size distribution.
The partic1e-size distribution data are usually reported as
Measurement cumulative undersize distribution andlor as density
distribution by volume. The symbol x is used to denote the
Precautions The instructions given in the instrument
partic1e size, which in turn is defined as the diameter of a
manual are followed:
volume-equivalent sphere. Q3(x) denotes the volume fraction
- never look into the direct path of the laser beam or its undersize at the partic1e size X . In a graphical representation,
reftections; x is plorted on the abscissa and the dependent variable Q3
- earth all instrument components to prevent ignition of on the ordinate. Most common characteristic values are
solvents or dust explosions; calculated from the partic1e-size distribution by interpolation.
- check the instrument set-up (e:g. warm-up, required The particle sizes at the undersize values of 10 per cent,
measuring range and lens, appropriate working distance, 50 per cent, and 90 per cent (denoted as X¡O, Xso, and X90
position of the detector, no direct bright daylight); respectively) are frequently used. Xso is also known as the
- in the case of wet dispersions, avoid air bubbles, median partic1e size. It is recognised that the symbol d is also
evaporation of liquid, schlieren or other inhomogeneities widely used to designate the partic1e size, thus the symbol x
in the dispersion; similarly, avoid improper mass-ftow may be replaced by d.
from the disperser or turbulent air-ftow in the case of dry Moreover, sufficient information must be documented about
the sample, the sample preparation, the dispersion
V -ASOS Appendix XVII Q 2014
conditions, and the cell type . As the results depend on the entity. Thus the entire measurement procedure is examined,
particular instrument, data analysis program, and optical including sample collection, sample dispersion, sample
model used, these details must also be documented. transport through the measuring zone, and the measurement
and deconvolution procedure. It is essential that the total
Control of the instrument perfonnance operational procedure is fully described.
Use the instrument according to the manufacturer's In general, unless otherwise specified in the individual
instructions and carry out the prescribed qualifications at an monograph, the response of a laser diffraction instrument is
appropriate frequency, according to the use of the instrument considered to meet the requirements if the XSO value does no t
and substances to be tested. deviate by more than 10 per cent from the range of values of
Calibration Laser diffraction systems, although assuming the reference material. If optionally the values at the sides of
idealised properties of the particles, are based on first the distribution are evaluated (e.g. X IO and X90), then these
principIes of laser light scanering. Thus, calibration in the values must not deviate by more than 15 per cent from the
strict sense is not required. However, it is still necessary to certified range of values. Below 1O ~lm, these values must be
confirm that the instrument is operating correctly. This can doubled.
be undertaken using any certified reference material that is NOTE: for calibration of the instrument, stricter requirements are
acceptable in industrial practice. The entire measurement laid down in the paragraph Calibration.
procedure is examined, including sample collection, sample
dispersion, sample transport through the measuring zone,
measurement, and the deconvolution procedure. It is
essential that the total operational procedure is fully Q. Characterisation of Crystalline and
described.
The preferred certified reference materials consist of spherical
Partially Crystalline Solids by X-ray
particles of a known distribution. They must be certified as Powder Diffraction (XRPD)
to the mass-percentage size distribution by an absolute (Ph. Eur. method 2.9.33)
technique, if available, and used in conjunction with an Every crystalline phase of a given substance produces a
agreed, detailed operation procedure. It is essential that the
characteristic X-ray diffraction pattem.
real and imaginary parts of the complex refractive index of
the material are indicated if the Mie theory is applied in data Diffraction pattems can be obtained from a randomly
analysis. The representation of the particle-size distribution oriented crystalline powder composed of crystallites or crystal
by volume will equal that of the distribution by mass, fragments of finite size. Essentially 3 types of information can
provided that the densiry of the particles is the same for all be derived from a powder diffraction pattem: angular
size fractions. position of diffraction lines (depending on geometry and size
of the unit cell); intensities of diffraction lines (depending
The response of a laser diffraction instrument is considered
mainly on atom type and arrangement, and particle
to meet the requirements if the mean value of XSO from at
orientation within the sample); and diffraction line profiles
least 3 independent measurements do es not deviate by more
(depending on instrumental resolution, crystallite size, strain
than 3 per cent from the certified range of values of the and specimen thickness).
certified reference material. The mean values for XIO and X90
must not deviate by more than 5 per cent from the certified Experiments giving angular positions and intensities of lines
range of values. Below 10 ¡.1m, these values must be doubled. can be used for applications such as qualitative phase analysis
(for example, identification of crystalline phases) and
Although the use of materials consisting of spherical particles
quantitative phase analysis of crystalline materials.
is preferable, non-spherical particles may also be employed.
An estimate of the amorphous and crystalline fractions 1 can
Preferably, these particles have certified or typical values from
also be made.
laser diffraction analyses performed according to an agreed,
detailed operating procedure. The use of reference values The X-ray powder diffraction (XRPD) method provides an
from methods other than laser diffraction may cause a advantage over other means of analysis in that it is usually
significant bias. The reason for this bias is that the different non-destructive in nature (specimen preparation is usually
principIes inherent in the various methods may lead to limited to grinding to ensure a randomly oriented sample).
different sphere-equivalent diameters for the same non- XRPD investigations can also be carried out under in situ
spherical particle. conditions on specimens expos.ed to non-ambient conditions,
such as low or high temperature and huinidity.
Although the use of certified reference materials is preferred,
other well-defined reference materials may also be employed. PrincipIe
They consist of substances of typical composition and
particle-size distribution for a specified class of substances. X-ray diffraction results from the interaction between X-rays
Their particle-size distribution has proven to be stable over and electron clouds of atoms. Depending on the atomic
time. The results must comply with previously determined arrangement, interferences arise from the scattered X-rays.
data, with the same precision and bias as for the certified These interferences are constructive when the path difference
reference material. between 2 diffracted X-ray waves differs by an integral
number of wavelengths. This selective condition is described
Qualification of the system In addition to the by the Bragg equation, also called Bragg's law (see Figure
calibration, the performance of the instrument must be 2.9.33.-1 ):
qualified at regular time intervals or as frequently as
appropriate. This can be undertaken using any suitable 1 There are many olher applications oi lhe X-ray powder diffraelion
reference material as mentioned in the previous paragraph. leehnique lhal can be applied lO eryslalline pharmaeeutical subslanees sueh
The qualification of the system is based on the concept that as: delermination oi eryslal structures, refinemem oi erystal Slntccures,
determination oi eryslallographie puriry oi Cl)!Slallil1e phases, eharacterisacion
the equipment, electronics, software and analytical operations
oi erystallographie texture, etc. These applicalions are nOl desm'bed in this
constitute an integral system, which can be evaluated as an ehapter.
2014 Appendix XVII Q V-A509
Figure 2.9.33.-1. - Diffraction of X-rays by a crystal according fo Bragg 's law
FormC
Form B
FormA
amorphous
i I i I I I i I I I i I i I i I i I ¡ I i I I I i 1 i I I I i I ¡ I i I i I i I i I ( I II j I ¡ I 11 ( I i
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
28 (A Cu) - Scale
Figure 2.9.33.-2. - X-ray powder diffraction patterns collecfed for 5 differenf solid phases of a subsfance
(the intensities are normalised)
V-AS! O Appendix XVII Q 2014
/
F
A. X-ray tube C. sample E. receiving slit G. detector receiving slit J. diffractometer circle
B. divergence slit D. anti-diffusion slit F. monochromator H. detector K. focusing circle
Figure 2.9.33.-3. - Geometric arrangement of the Bragg-Brentano parafocusing geometry
theoretical intensities depend on the crystallographic unit cell

content (nature and positions of atoms), and the line profiles
The wavelength le of the X-rays is of the same order of on the perfection and extent of the crystallattice. Under
magnitude as the distance between successive crystal lattice these conditions the diffraction peak has a finite intensity
planes, or dhkl (also called 'd-spacings'). 8hkl is the angle arising from atomic arrangement, type of atoms, thermal
between the incident ray and the family of lattice planes, and motion and structural imperfections, as well as from
sin8 hkl is inversely proportional to the distance between instrument characteristics.
successive crystal planes or d-spacings. The intensity is dependent upon many factors such as
The direction and spacing of the planes with reference to the structure factor, temperature factor, crystallinity, polarisation
unit cell axes are defined by the Miller indices {hkl}. These factor, multiplicity and Lorentz factor.
indices are the reciprocals, reduced to the next-lower integer, The main characteristics of diffraction line profiles are 28
of the intercepts that aplane makes with the unit cell axes. position, peak height, peak area and shape (characterised by,
The unit cell dimensions are given by the spacings a, b and e for example, peak width or asyrnmetry, analytical function,
and the angles between them, Ci, ~, and y. empirical representation). An example of the type of powder
The interplanar spacing for a specified set of parallel hkl patterns obtained for 5 different salid phases of a substance
planes is denoted by dhkl . Each such family of planes may are shown in Figure 2.9.33.-2.
show higher orders of diffraction where the d values for the In addition to the diffraction peaks, an X-ray diffraction
related families of planes nh, nk, ni are diminished by the experiment also genera tes a more-or-less uniform
factor l/n (n being an integer: 2, 3, 4, etc.). background, upon which the peaks are superimposed.
Every set of planes throughout a crystal has a corresponding Besides specimen preparation, other factors contribute to the
Bragg diffraction angle, 8hkl, associated with it (for a specific background, for instance the sample holder, diffuse scattering
wavelength le) . from air and equipment, other instrumental parameters such
A 'p owder specimen is assumed to be polycrystalline so that as detector noise, general radiation from the X-ray tube, etc.
at any angle 8hkl there are always crystallites in an orientation The peak-to-background ratio can be increased by
allowing difftaction according to Bragg's law2 . For a given minimising background and by choosing prolonged exposure
X-ray wavelength, the positions of the diffraction peaks (also times.
referred to as 'lines', 'refiections' or 'Bragg refiections') are
characteristic of the crystallattice (d-spacings), their Instrument
Instrument set-up X-ray diffraction experiments are
2 An 'ideal' powder jor diffraction experiments consists oj a large number oj
usually performed using powder diffractometers or powder
small, randomly oriemed spherical crysrallites (coheremly dijfracting
crystalline domains). Jj Ihis number is sufficiemly large, ¡here are always
cameras.
enough cryswllites in any diffracting orientadon /O give reproducible A powder diffractometer generally comprises 5 main parts: an
diffraction pallems. X-ray source; incident beam optics, which may perform
2014 Appendix XVII Q V-AS11
monochromatisation, filtering, coIlimation and/or focusing of experiments. The principal radiation sources utilised for
the beam; a goniometer; diffraction beam optics, which may X-ray diffraction are vacuum tubes utilising copper,
perform monochromatisation, filtering, collimation and molybdenurn, iron, cobalt or chromium as ano des; copper,
focusing or parallelising of the beam; and a detector. Data- molybdenum or cobalt X-rays are employed most commonly
coIlection and data-processing systems are also required and for organic substances (the use of cobalt anodes can be
are generally included in current diffraction measurement especially preferred to separate distinct X-ray lines) .
equipment. The choice of radiation to be used depends on the
Depending on the type of analysis to be performed (phase absorption characteristics of the specimen and possible
identification, quantitative analysis, lattice parameters fiuorescence by atoms present in the specimen.
determination, etc.), different XRPD instrument The wavelengths used in powder diffraction generally
configurations and performance levels are required. correspond to the K:x radiation from the ano de.
The simplest instruments used to measure XRPD pattems Consequently, it is advantageous to make the X-ray beam
are powder cameras. The replacement of photographic film 'monochromatic' by eliminating all the other components of
as the detection method by photon detectors has led to the the emission spectrum. This can be partly obtained using K p
design of diffractometers in which the geometric arrangement filters, i.e. metal filters selected as having an absorption edge
of the optics is not truly focusing but parafocusing, such as in between the K:x and Kp wavelengths emitted by the tube.
the Bragg-Brentano geometry. The Bragg-Brentano Such a filter is usually inserted between the X-ray tube and
parafocusing configuration is currently the most widely used the specimen. Another, more-and-more-commonly used way
and is therefore briefiy described here. to obtain a monochromatic X-ray beam is via a large
A given instrument may provide a horizontal or vertical 8/28 monochromator crystal (usually referred to as a
geometry or a vertical 8/8 geometry. For both geometries, the 'monochromator') . This crystal is placed before or behind
incident X-ray beam forms an angle 8 with the specimen the specimen and diffracts the different characteristic peaks
surface plane and the diffracted X-ray beam forms an angle of the X-ray beam (i.e. K:x and Kp) at different angles, so
28 with the direction of tlle incident X-ray beam (an angle 8 that only one of them may be selected to enter into the
with the specimen surface plane) . The basic geometric detector. It is even possible to separate K:xl and K:x2
arrangement is represented in Figure 2.9.33.-3. radiations by using a specialised monochromator.
The divergent beam of radiation from the X-ray tube (the Unfortunately, the gain in getting a monochromatic beam by
so-called 'primary beam') passes through the parallel plate using a filter or a monochromator is counteracted by a loss in
coIlimators and a divergence slit assembly and illuminates the intensity. Another way of separating K:x and K p wavelengths
fiat surface of the specimen. AII the rays diffracted by is by using curved X-rays mirrors that can simultaneously
suitably oriented crystallites in the specimen at an angle 28 monochromate and focus or parallelise the X-ray beam.
converge to a line at the receiving slit. A second set of RADIA TJON PROTECTION. Exposure of any parl of lhe
parallel plate collimators and a scatter slit may be placed human body 10 X-rays can be injurious 10 heallh. JI is lherefore
either behind or before the receiving slit. The axes of the line essenliallhat whenever X-ray equipmenl is used, adequate
focus and of the receiving slit are at equal distances from the precautions are taken to protect the operalOr and any other person
axis of the goniometer. The X-ray quanta are counted by a in the vicinity. R ecommended practice for radiation protection as
radiation detector, usually a scintillation counter, a sealed-gas well as limits for the levels of X -radiation exposure are those
proportional counter, or a position-sensitive solid-state established by national legislation in each country. Jf there are no
detector such as imaging plate or CCD detector. official regulations or recommendations in a COUnlry, the latest
The receiving slit assembly and the detector are coupled recommendations of the Jnlemational Commission on Radiological
together and move tangentially to the focusing circ1e. Protection should be applied.
For 8/28 scans the goniometer rotates the specimen about the
same axis as that of the detector, but at half the rotational Specimen preparation and mounting
speed, in a 8/28 motion. The surface of the specimen thus The preparation of the powdered material and mounting of
remains tangential to the focusing circ1e. The parallel plate the specimen in a suitable holder are critical steps in many
coIlimator limits the axial divergence of the beam and hence analytical methods, and are particularly so for XRPD
partially controls the shape of the diffracted line profile. analysis, since they can greatly affect the quality of the data
A diffractometer may also be used in transmission mode. to be collected3 . The main sources of error due to specimen
The advantage with this technology is to les sen the effects preparation and mounting are briefiy discussed here for
due to preferred orientation. A capillary of about 0.5-2 mm instruments in Bragg-Brentano parafocusing geometry.
thickness can also be used for small sample amounts. Specimen preparation
X-ray radiation In the laboratory, X-rays are obtained by In general, the morphology of many crystaIline partic1es tends
bombarding a metal anode with electrons emitted by the to give a specimen that exhibits sorne degree of preferred
thermionic effect and accelerated in a strong electric field orientation in the specimen holder. This is particularly
(using a high-voltage generator). Most of the kinetic energy evident for needle-like or plate-like crystals when size
of the electrons is converted to heat, which limits the power reduction yields finer needles or platelets. Preferred
of the tubes and requires efficient ano de cooling. A 20- to orientation in the specimen infiuences the intensities of
30-fold increase in brilliance can be obtained using rotating various refiections, so that sorne are more intense and others
ano des and by using X-ray optics. Altematively, X-ray are less intense, compared to what would be expected from a
photons may be produced in a large-scale facility completely random specimen. Several techniques can be
(synchrotron) . employed to improve randomness in the orientation of
The spectrum emitted by an X-ray tube operating at crystaIlites (and therefore to rninimise preferred orientation),
sufficient voltage consists of a continuous background of but further reduction of partic1e size is often the best and
polychromatic radiation and additional characteristic
radiation that depends on the type of anode. Only this 3 Similarly, changes in the specimen can occur during data collecnon in ¡he
characteristic radiation is used in X-ray diffraction case oi a non-equilibrium specimen (temperature, hwnidity).
V-A512 Appendix XVII Q 2014
simplest approach. The optimum number of crystallites investigation. Therefore, the different components of the
depends on the diffractometer geometry, the required diffractometer must be carefully adjusted (optical and
resolution and the specimen attenuation of the X-ray beam. mechanical systems, etc.) 10 minimise adequately systematic
In sorne cases, particle sizes as large as 50 ¡¡m will provide errors, while optimising the intensities received by the
satisfactory results in phase identification. However, excessive detector. The search for maximum intensity and maximum
milling (crystallite sizes less than approximately 0.5 ¡lm) may resolution is always antagonistic when aligning a
cause line broadening and significant changes to the sample diffrac1Ometer. Hence, the best compromise must be sought
itself such as: whilst performing the alignment procedure. There are many
- specimen contamination by particles abraded from the different configurations and each supplier's equipment
milling instruments (mortar, pestle, balls, etc.); requires specific alignment procedures.
- reduced degree of crystallinity; The overall diffrac10meter performance must be tested and
- solid-state transition to another polymorph; moni1Ored periodically using suitable certified reference
materials. Depending on the type of analysis, other well-
- chemical decomposition; defined reference materials may also be employed, although
- introduction of internal stress; the use of certified reference materials is preferred.
- solid-state reactions.
Therefore, it is advisable to compare the diffraction pattern Qualitative phase analysis (Identification of phases)
of the non-ground specimen with that corresponding to a The identification of the phase composition of an unknown
specimen of smaller particle size (e.g. a milled specimen). sample by XRPD is usually based on the visual or computer-
If the XRPD pattern obtained is of adequate quality assisted comparison of a portion of its XRPD pattern 10 the
considering its intended use, then grinding may not be experimental or calculated pattern of a reference material.
required. Ideally, these reference patterns are collected on well-
It should be noted that if a sample contains more than one characterised single-phase specimens. This approach makes it
phase and if sieving is used to isolate particles to a specific possible in most cases 10 identify a crystalline substance by its
size, the initial composition may be altered. 28 diffraction angles or d-spacings and by its relative
intensities. The computer-aided comparison of the diffraction
Specimen mounting pattern of the unknown sample 10 the comparison data can
EfIect of specimen displacement A specimen surface be based either on a more-or-Iess extended 28-range of the
that is offset by D with reference 10 the diffractometer whole diffraction pattern or on a set of reduced data derived
rotation axis causes systematic errors that are very difficult to from the pattern. For example, the list of d-spacings and
avoid entirely; for the refiection mode, this results in absolute normalised intensities (Inorm), a so-called (d, Inorm)-list
D·cos8 shifts 4 in 28 positions (typically of the order of 0.01 ° extracted from the pattem, is the crystallographic fingerprint
in 28 at low angles (cos8 '" 1) for a displacement of the material, and can be compared 10 (d, Ino~ -lists of
D = 15 ¡.lm) and asymmetric broadening of the profile single-phase samples compiled in databases.
towards low 28 values. Use of an appropriate internal For most organic crystals, when using Cu Ka radiation, it is
standard allows the detection and correction of this effect appropriate to record the diffraction pattern in a 28-range
simultaneously with that arising from specimen transparency. from as near 0° as possible to at least 40°. The agreement in
This is by far the largest source of errors in data collected on the 28-diffraction angles between specimen and reference is
well-aligned diffractometers. within 0.2° for the same crystal form, while relative intensities
EfIect of specimen thickness and transparency When between specimen and reference may vary considerably due
the XRPD method in refiection mode is applied, it is often to preferred orientation effects. By their very nature, variable
preferable 10 work with specimens of 'infinite thickness'. hydrates and solvates are recognised to have varying unit cell
To minimise the transparency effect, it is advisable 10 use a dimensions and as such shifting occurs in peak positions of
non-diffracting substrate (zero background holder), for the measured XRPD 'patterns for these materials. In these
example a plate of single crystalline silicon cut parallel to the unique materials, variance in 28-positions of greater than 0.2°
510 lattice planes 5 • One advantage of the transmission mode is not unexpected. As such, peak position variances such as
is that problems with sample height and specimen 0.2° are not applicable 10 these materials. For other types of
transparency are less important. The use of an appropriate samples (e.g. inorganic salts), it may be necessary to extend
internal standard allows the detection and correction of this the 28-region scanned to well beyond 40°. It is generally
effect simultaneously with that arising from specimen sufficient 10 sean past the 10 strongest refiections identified
displacement. in single phase XRPD database files.
It is sometimes difficult or even impossible 10 identify phases
Control of the instrument perfonnance
in the following cases:
Goniometers and the corresponding incident and diffracted
- non-crystallised or amorphous substances;
X-ray beam optics have many mechanical parts that need
adjustment. The degree of alignment or misalignment - the components to be identified are present in low mass
directly infiuences the quality of the results of an XRPD fractions of the analyte amounts (generally less than
10 per cent m/m);
4 Note that a gonionzeter zero alignnzent shift would result in constant shift
- pronounced preferred orientation effects;
on all observed 2e-line positions, in other words, the whole diffraction partern - the phase has not been filed in the database used;
is in this case translated by an offsec of ZO in 2•. - formation of solid solutions;
5 In che case of a ¡hin specimen with low artenuation, accurate nzeasurenzents
of line posirionf can be macft wi¡h focusing diffraccometer configurations in - presence of disordered structures that alter the unit cell;
eicher cransmission or reflection geometly. Accurace measurements of line - the specimen comprises too many phases;
positions on specimens wich low artenuation are preferably made using - presence of lattice deformations;
diffracrometers wich paraUe! beam opcics. This helps 10 reduce che effects of
specimen chickness. - structural similarity of different phases;
2014 Appendix XVII R V -A513
Quantitative phase analysis to prepare an intensity-versus-concentration plot in which the

If the sample under investigation is a mixture of 2 or more negative x intercept is the concentration of the phase a in the
known phases, of which not more than 1 is amorphous, the original sample.
percentage (by volume or by mass) of each crystalline phase Estimate of the amorphous and crystalline fractions
and of the amorphous phase can, in many cases, be
determined. Quantitative phase analysis can be based on the In a mixture of crystalline and amorphous phases, the
integrated intensities, on the peak heights of several crystalline and amorphous fractions can be estimated in
individual diffraction lines 6, or on the full pattern. These several ways. The choice of the method used depends on the
integrated intensities, peak heights or full-pattern data points nature of the sample:
are compared to the corresponding values of reference -- if the sample consists of crystalline fractions and an
materials. These reference materials shall be single-phase or a amorphous fraction of different chemical compositions,
mixture of known phases. The difficulties encountered during the amounts of each of the individual crystalline phases
quantitative analysis are due to specimen preparation (the may be estimated using appropriate standard substances
accuracy and precision of the results require in particular as described aboye; the amorphous fraction is then
homogeneity of all phases and a suitable particle size deduced indirectly by subtraction;
distribution in each phase) and to matrix effects. -- if the sample consists of one amorphous and one
In favourable cases, amounts of crystalline phases as small as crystalline fraction, either as a l-phase or a 2-phase
10 per cent may be determined in solid matrices. mixture, with the same elemental composition, the
Polymorphic samples amount of the crystalline phase ('the degree of
crystallinity') can be estimated by measuring 3 areas of the
For a sample composed of 2 polymorphic phases a and b, the
diffractogram:
following expression may be used to quantify the fraction Fa
ofphase a: A total area of the peaks arising from diffraction from the
crystalline fraction of the sample;
l B total area below area A;
Fa = ----~--~~
l + K (hila) C - background area (due to air scattering, fluorescence,
equipment, etc) .
The fraction is derived by measuring the intensity ratio When these areas have been measured, the degree of
between the 2 phases, knowing the value of the constant K. crystallinity can be roughly estimated using the following
K is the ratio of the absolute intensities of the 2 pure formula:
polymorphic phases loa/loboIts value can be determined by
measuring standard samples. % crystallinity = IOOAI (A +B - C)
Methods using a standard
It is noteworthy that this method does not yield absolute
The most commonly used methods for quantitative analysis
degree-of-crystallinity values and hence is generally used for
are :
comparative purposes only.
-- the 'external standard method';
More sophisticated methods are also available, such as the
-- the 'internal standard method'; Ruland method.
-- the 'spiking method' (often also called the 'standard
addition method'). Single crystal structure
The 'extemal standard method' is the most general method In general, the determination of crystal structures is
and consists of comparing the X -ray diffraction pattern of the performed from X-l;ay diffraction data obtained using single
mixture, or the respective line intensities, with those crystals. However, crystal structure analysis of organic crystals
measured in a reference mixture or with the theoretical is a challenging task, since the lattice parameters are
intensities of a structural model, if it is fully known. comparatively large, the symmetry is low and the scattering
To limit errors due to matrix effects, an internal reference properties are normally very low.
material with crystallite size and X-ray absorption coefficient For any given crystalline form of a substance, knowledge of
comparable to those of the components of the sample, and the crystal structure allows the calculation of the
with a diffraction pattern that does not overlap at all that of corresponding XRPD pattern, thereby providing a 'preferred-
the sample to be analysed, can be used. A known quantity of orientation-free' reference XRPD pattern, which may be used
this reference material is added to the sample to be analysed for phase identification.
and to each of the reference mixtures . Under these
conditions, a linear relationship between line intensity and
concentration exists. This application, called the 'internal R. Porosity and Pore-size Distribution of
standard method', requires a precise measurement of
diffraction intensities . . Solids by Mercury Porosimetry
In the 'spiking method' (or 'standard addition method'), (Ph. Eur. method 2.9.32)
sorne of the pure phase a is added to the mixture containing
the ¡mknown concentration of a. Multiple additions are made Introduction
In general, different types of pores may be pictured as
apertures, channels or cavities within a solid body, or as
6 Jf che crystal S11'Uctures of all componems are k11own, che Reitveld mechod
space (i.e. interstices or voids) between solid particles in a
can be used to quantify them wich good accuracy. Jf che crystal scruaures of bed, compact or aggregate. Porosity is a term that is often
che components are 110C known, the Pawley or least squares mechods can be used to indicate the porous nature of solid material, and is
used. more precisely defined as the ratio of the volume of
V -A514 Appendix XVII R 2014
accessible pores and voids to the total volume occupied by a applied pressure. The measurement ineludes only those pores
given amount of the solido In addition to the accessible pores, into which mercury can penetrate at the pressure applied,
a solid may contain elosed pores, which are isolated from the A non-wetting liquid penetra tes into a porous system only
external surface and into which fiuids are not able to under pressure. The pressure to be applied is in inverse
penetrate . The characterisation of closed pores, i.e. cavities proportion to the inner diameter of the pore aperture. In the
with no access to an external surface, is not covered in this case of cylindrical pores, the correlation between pore
chapter. diameter and pressure is given by the Washburn equation:
Porous materials may take the form of fine or coarse
powders, compacts, extrudates, sheets or monoliths. Their
characterisation usually involves the determination of the
total pore volume or porosity as well as the pore-size
distribution. dp pore diameter, in metres;
It is well established that the performance of a porous solid (J surface tension, in newtons per metre;
(e.g. its strength, reactivity, permeability or adsorbent power) e contact angle of mercury on the sample, in degrees;
is dependent upon its pore structure. Many different p applied pressure, in pascals.
methods have been developed for the characterisation of pore
structure. In view of the complexity of most porous solids, it Apparatus
is not surprising to find that the results obtained are not The sample holder, referred to as penetrometer or
always in agreement and that no single technique can be dilatometer, has a calibrated capillary tube, through which
relied upon to provide a complete picture of the pore the sample can be evacuated and through which mercury can
structure. The choice of the most appropriate method enter. The capillary rube is anached to a wider tube in which
depends on the application of the porous solid, its chemical the test sample is placed. The change in the volume of
and physical nature and the range of pore-size . mercury intruded is usually measured by the change in
This chapter provides guidance for measurement of porosity capacitance between the mercury column in the capillary
and pore-size distribution by mercury porosimetry. It is a tube and a metal sleeve around the outside of the capillary
comparative test, usually destructive, in which the volume of tube. If precise measurements are required the expected total
mercury penetrating a pore or void is determined as a void and pore volume of the sample should be between
function of an applied hydrostatic pressure, which can be 20 per cent and 90 per cent of the internal volume of the
related to a pore diameter. Other information such as pore capillary tube, Since different materials exhibit a wide range
shape and inter-connectivity, the internal and external surface of open porosities, a number of penetrometers with different
area, powder granulometry, bulk and tapped density could capillary tube diarneters and sample volumes may be
also be inferred from volume-pressure curves; however, these required. A typica! set-up for a mercury porosimeter
aspects of the technique do not fall under the scope of this instrument is given in Figure 2,9.32.-l. The porosirneter may
chapter. have separate ports for high- and low-pressure operation, or
Practica! considerations presently limit the maximum applied the low-pressure measurement may be carried out on a
absolute pressure reached by sorne equipment to about separate unit.
400 MPa, corresponding to a minimum equivalent pore The pressure range is typically 4-300 kPa for low-pressure
diameter of approximately 0.003 ~m. The maximum operation and aboye 300 kPa for high-pressure operation,
diameter will be limited for samples having a significant depending on the design of the particular apparatus and on
depth due to the difference in hydrostatic head of mercury the intended use.
from the top to the bonom of the sample. For most purposes
this limit may be regarded as 400 ~m. Method
Inter-partiele and intra-particle porosity can be determined, Sample preparation
but the method does not distinguish between these porosities The sarnple is pre-treated to remove adsorbed material that
where they co-exist. ' can obscure its accessible porosity, for example by heating
The method is suitable for the study of most porous and/or evacuation, or by fiowing inert gas. It may be possible
rnaterials, Samples that amalgamate with mercury, such as to passivate the suliace of wettable or amalgam-forming
certain metal s, may be unsuitable for this technique or may solids, for example by producing a thin layer of oxide, or by
require a preliminary passivation, Other materials may coating with stearate.
deform or compact under the applied pressure, In sorne cases The sample of the pre-treated solid is weighed and
it may be possible to apply sample-compressibility corrections transferred to the penetrometer. The pore system of the
and useful comparative data may still be obtained, sample is then degassed in a vacuum to a maximum residual
Mercury porosimetry is considered to be comparative, as for pressure of 7 Pa.
most porous media a theory is not available to allow an Filling the penetrometer with mercury
absolute ca1culation of results of pore-size distribution. The mercury used is of analytical quality. Overlay the sample
Therefore this technique is mainly recommended for with mercury under vacuum. The vacuum is required to
development studies. ensure the transfer of mercury from the reservoir to the
Mercury is toxico Appropriate precautions must be observed penetrometer. In a filled penetrometer the filling pressure
to safeguard the health of the operator and others working in comprises the applied pressure plus the pressure contribution
the area. Waste material must also be disposed of in a created by the head of mercury contacting the sample.
suitable manner, according to local regulations. A typical filling pressure would be about 4 kPa.
The hydrostatic pressure of the mercury over the sample may
PrincipIe be minimised by filling the penetrometer in the horizontal
The technique is based on the measurement of the mercury position.
volume intruded into a porous solid as a function of the
2014 Appendix XVII R V -A515
8-,r----1
11 ______
E
1.....-----1 Q
H
111..----," 1 1
1
1 "
J
!B 1
1
1
K
L
LlJ 1
1
1
1
M
1
N 1
e
-8
A. Low-pressure hydraulic fluid E. High-pressure hydraulic fluid J. Penetration volume N. Sample
reservo ir reservo ir indicator
B. Hydraulic pump F. Vacuum pump with gauge K. Capillary tube
C. Pressure multiplier G. Mercury reservoir L. High-pressure chamber
D. Pressure transducer H. Oil M. Mercury
Figure 2.9.32.-1. - Example of the set-up of a mercury porosimeter instrument
Low-pressure measurement 1?300r---------------------------------,

E
Admit air or nitro gen in a controlled manner to increase the .s 240
Q)
pressure either in stages corresponding to the particular pore E
:J
sizes of interest, or continuously at a slow rateo ~ 180
()
The concomitant change in the length of the mercury '"
'ü
column in the capillary tube is recorded. When the 1i 120
maximum required pressure has been reached, retum to '"
Q)
.<:
1ií 60
atmospheric pressure. '3
E
High-pressure measurement c3 o 1~0~·'....=====1;;;0;:O
::..-------1::":0::"--------~10'
After measurement at low pressure, the penetro meter filled Pressure (MPa)
with mercury is transferred to the high-pressure port or unit
of the instrument and overlaid with hydraulic fluid. Mercury Figure 2.9.32.-2. - Volume-pressure curve as semilogarithmic
is intruded into the pore system via the hydraulic fluid. plot
Increase the pressure in the system to the maximum pressure
reached in the low-pressure measurement 'and record the
intrusion volume at this pressure, since subsequent intrusion Reporting of results
volumes are calculated from this initial volume. Increase the The pressure 'readings can be converted to pore diameters by
pressure either in stages corresponding to the particular pore means of the Washbum equation or by another model.
sizes of interest, or continuously at a slow rateo The fall in The surface tension of mercury (G) depends not only on the
the mercury column is measured up to the maximum temperarure, but also, in the case of markedly curved
required pressure. If required the pressure may be decreased surfaces areas, on the radius of curvarure . In general, values
either in stages or continuously at a slow rate to determine between 0.41 N·m- 1 and 0.52 N·m- 1 are measured at room
the mercury extrusion curve. temperature. If the value is not known, () = 0.48 N·m- 1 can
Corrections are made to take account of changes in the be used.
volume of the mercury, the penetrometer and other The contact angle of mercury (8) in most cases is more than
components of the volume detector system under elevated 90°. It may be determined using a contact angle instrumento
pressure. The extent of the corrections may be determined If the value is not known, 8 = 130 D can be used. The values
by means of blank measurements under the same conditions. of contact angle and surface tension and the model used in
An experimentally determined volume-pressure curve is the calculation are reported.
shown in Figure 2.9 .32.-2. Visualisation of the data can be done with several types of
graphs. Frequently, in a graphical representation the pore
diameter is plotted on the abscissa and the intruded volume
per sample mass on the ordinate to give the pore-size
distribution. It is appropriate here to choose a logarithmic
scale for the abscissa (see Figure 2.9.32 .-3). The spaces
between the partic1es of the solid sample are inc1uded as
pores in the calculation. If the pores differ in size from the
V-A516 Appendix XVII S 2014
voids, the latter can be separated by choosing the appropriate The bulk density of a powder is determined either by
pore-size range. measuring the volume of a known mass of powder sample,
which may have been passed through a sieve, in a graduated
~ 300
M
cylinder (Method 1), or by measuring the mas s of a known
E volume of powder that has been passed through a volumeter
.s 240
al
into a cup (Method 2) or has been introduced into a
E measuring vessel (Method 3).
:::l
~ 180 >.
u Methods 1 and 3 are favoured.
ü e
¡¡:: al
'13 :::l
¡ii 120 CT Method 1: Measurement in a graduated cylinder
~
<f)
al
lL Procedure Pass a quantity of powder sufficient to
> complete the test through a sieve with apertures greater than
~ 60
:; or equal to 1.0 mm, if necessary, to break up agglomerates
E
O
:::l
O
that may have formed during storage; this must be done
10' gently to avoid changing the nature of the material. Into a
Pore diameter (nm) dry, graduated, 250 mL cylinder (readable to 2 mL), gently
introduce, without compacting, approximately 100 g (m) of
Figure 2.9.32.-3. - Pore-size distribution as semilogarithmic the test sample weighed with 0.1 per cent accuracy.
plots ofthe cumulative and the normalised density distribution If necessary, carefully level the powder without compacting,
and read the unsettled apparent volume (Vo) to the nearest
Extrusion curves may not be used for calculating the pore- graduated unit. Calculate the bulk density in grams per
size distribution (for hysteresis, see Figure 2.9.32.-2), because millilitre using the formula m/Vo. Generally, replicate
an intruded part of the mercury always remains in the pore determinations are desirable for the determination of this
system. The retention ratio may however be useful for the property.
qualitative characterisation of pores that are only accessible If the powder density is too low or too high, such that the
via narrow openings ('ink-bottle pores'). test sample has an untapped apparent volume of more than
Most common characteristic values, such as the total 250 mL or less than 150 mL, it is not possible to use 100 g
intruded specific volume and the mean and median pore of powder sample. In this case, a different amount of powder
diameters, are calculated from the pore-size distribution. is selected as the test sample, such that its untapped apparent
Moreover, sufficient information must be documented about volume is between 150 mL and 250 mL (apparent volume
the sample, the sample preparation, the evacuation greater than or equal to 60 per cent of the total volume of
conditions and the instrument used. the cylinder); the mass of the test sample is specified in the
expression of results.
Control of instrument perfonnance For test samples having an apparent volume between 50 mL
As mercury porosimetry is considered to be used as a and 100 mL, a 100 mL cylinder readable to 1 mL can be
comparative test, no details are given in this chapter. used; the volume of the cylinder is specified in the expression
However, it is recommended that a stable comparison of results.
material is tested on a regular basis to monitor instrument
calibration and performance. Method 2: Measurement in a volumeter
Apparatus The apparatus (Figure 2.9.34.-1) consists of a
top funnel fitted with a 1.0 mm sieve, mounted over a baffle
box containing 4 glass baffles over which the powder slides
S. Bulk Density and Tapped Density of and bounces as it passes. At the bottom of the baffle box is a
funnel that collects the powder and allows it to pour into a
Powders cup mounted directly below it. The cup may be cylindrical
(Ph. Eur. method 2.9.34) (25 .00 ± 0.05 mL volume with an internal diameter of
30.00 ± 2.00 mm) or cubical (16.39 ± 0.20 mL volume
Bulk density with internal dimensions of 25.400 ± 0.076 mm).
The bulk density of a powder is the ratio of the mass of an Procedure AlIow an excess of powder to fiow through the
untapped powder sample to its volume, including the apparatus into the sample receiving cup until it overflows,
contribution of the interparticulate void volume. Hence, the using a minimum of 25 cm 3 of powder with the cubical cup
bulk density depends on both the density of powder particles and 35 cm 3 of powder with the cylindrical cup. Carefully,
and the spatial arrangement of particles in the powder bed. scrape excess powder from the top of the cup by smoothly
The bulk density is expressed in grams per millilitre despite ínoving the edge of the blade of a spatula perpendicular to
the International Unit being kilogram per cubic metre and in contact with the top surface of the cup, taking care to
(1 g/mL = 1000 kg/m 3), because the measurements are keep the spatula perpendicular to prevent packing or removal
made using cylinders. It may also be expressed in grams per of powder from the cup. Remove any material from the side
cubic centimetre. of the cup and determine the mass (M) of the powder to the
The bulking properties of a powder are dependent upon the nearest 0.1 per cent. Calculate the bulk density in grams per
preparation, treatment and storage of the sample, i.e. how it millilitre using the formula M/Vo (where Vo is the volume of
has been handled. The particles can be packed to have a the cup) and record the average of 3 determinations using 3
range of bulk densities and, moreover, the slightest different powder samples.
disturbance of the powder bed may result in a changed bulk
density. Thus, the bulk density of a powder is often very
difficult to measure with good reproducibility and, in
reporting the results, it is essential to specify how the
determination was made.
2014 Appendix XVII S V -AS 17
A
record the average of 3 determinations using 3 different
powder samples.
\J V
B
\\/ U
-
Tapped density
The tapped density is an increased bulk density attained after
mechanically tapping a receptacle containing the powder
sample.
The tapped density is obtained by mechanically tapping a
e graduated measuring cylinder or vessel containing the powder
sample. After observing the initial powder volume or mass,
the measuring cylinder or vessel is mechanically tapped, and
volume or mass readings are taken untillittle further volume
or mass change is observed. The mechanical tapping is
/
f--
achieved by raising the cylinder or vessel and allowing it to
\_\1
drop, under its own mass, a specified distance by one of
3 methods as described below. Devices that rotate the
D cylinder or vessel during tapping may be preferred to
minimise any possible separation of the mas s during tapping
down.
Method 1
Apparatus The apparatus (Figure 2.9.34.-3) consists of
E the following:
G
"\ - a 250 mL graduated cylinder (readable to 2 mL) with a
mas s of 220 ± 44 g;
\ / - a settling apparatus capable of producing, per minute,
'---'
either nominally 250 ± 15 taps from a height of
3 ± 0.2 mm, or nominally 300 ± 15 taps from a height
F
(
n 1
of 14 ± 2 mm. The support for the graduated cylinder,
with its holder, has a mas s of 450 ± 10 g.
Procedure Proceed as described aboye for the
determination of the bulk volume (Vo). Secure the cylinder
A. 1.0 mm sieve E. glass baffle in the support. Carry out 10, 500 and 1250 taps on the
same powder sample and read the corresponding volumes
B. powder funnel F. cup
VIO' V 500 and V I250 to the nearest graduated unit. If the
C. loading funnel G. stand difference between V 500 and V I250 is less than or equal to
D. baffle box
2 mL, V 1250 is the tapped volume. If the difference between
Vsoo and V1250 exceeds 2 mL, repeat in increments of, for
Figure 2.9.34.-1. - Volumeter example, 1250 taps, until the difference between successive
measurements is less than or equal to 2 mL. Fewer taps may
be appropriate for sorne powders, when validated. Calculate
Method 3: Measurernent in a vessel
the tapped density in grams per millilirre using the formula
Apparatus The apparatus consists of a 100 mL cylindrical m/V¡ (where V¡ is the final tapped volume). Generally,
vessel of stainless steel with dimensions as specified in Figure replicate determinations are desirable for the determination
,
2.9.34.-2. of this property. Specify the drop height with the results.
--54.0 ----+ __ 57.0----+ If it is not possible to use a 100 g test sample, use a reduced
amount and a suitable 100 mL graduated cylinder (readable
o
50.5 r [50.5] r to 1 mL) weighing 130 ± 16 g and mounted on a support
weighing 240 ± 12 g. The modified test conditions are
52.0 50.0
specified in the expression of the results .
2:0 J 10;0 540 J Method2
Procedure Proceed as directed under Method 1 except
that the mechanical tester provides a fixed drop of
Figure 2.9.34.-2. - Measuring vessel (left) and cap (right) 3 ± 0.2 mm at a nominal rate of 250 taps per minute.
Method 3
Procedure Pass a quantity of powder sufficient to Procedure Proceed as described under Method 3 for
complete the test through a 1.0 mm sieve, if necessary, to measuring the bulk density, using the measuring vessel
break up agglomerates that may have formed during storage, equipped with the cap shown in Figure 2.9 .34.-2 .
and allow the obtained sample to fiow freely into the The measuring vessel with the cap is lifted 50-60 times per
measuring vessel until it overfiows. Carefully scrape the minute by the use of a suitable tapped density testero Carry
excess powder from the top of the vessel as described under out 200 taps, remove the cap and carefully scrape excess
Method 2. Determine the mass (Mo) of the powder to the powder from the top of the measuring vessel as described
nearest 0.1 per cent by subrracting the previously determined under Method 3 for measuring the bulk density. Repeat the
mass of the empty measuring vessel. Calculate the bulk procedure using 400 taps. If the difference between the 2
density in grams per millilirre using the formula Mol 100 and masses obtained after 200 and 400 taps exceeds 2 per cent,
V-A518 Appendix XVII T 2014
I E
-:¡:- E
-' L{)
..:t:.. E E C"l
o E .E C"l
L{)
o 01 e
N o 'Qi ro
...±... 1:: N .c E
ro
Graduated eylinder
-±- a. ero 2 ~
o
al E
"O
~
:±: 10 r/l
r/l
E
'O
-*-
T
::J
"O
~
~
'O
e
Cylinder --.- C) e
support ...:¡:...
Anvil
This dimension is su eh
that the drop CD meets
,...J--¡-;t;;======~speeifieations and that, at
t the lowest point of the eam,
(
the eylinder support is sitting
freely on the upper part of
the anvil.
Cam
Figure 2.9.34.-3. - Settling device for powder samples

repeat the test using 200 additional taps unril the difference Compressibility index:
between successive measuremenrs is less than 2 per cent.
Calculate the tapped density in grams per millilitre using the 100 (Vo - V¡)
formula MI /100 (where MI is the mas s of powder in the Vo
measuring vessel). Record the average of 3 determinations
using 3 differenr powder samples. The test conditions, Vo = unsertled apparenr volume;
including tapping height, are specified in the expression of V¡ = final tapped volurne.
the results. Hausner Ratio :
Measutes of powder compressibility Vo
Because the interparticulate interactions infiuencing the V¡
bulking properties of a powder are also the inreractions that
inrerfere with powder fiow, a comparison of the bulk and Depending on the material, the compressibility índex can be
tapped densities can give a measure of the relative determined using ViO instead of Vo. If VIO is used, it is
importance of these interactions in a given powder. Such a clearly stated with the results.
comparison is often used as an index of the ability of the
powder 10 fiow, for example the cbmpressibility index or the
H ausner ratio.
The compressibility index and Hausner ratio are mea sures of T. Wettability of Porous Solids Including
the propensity of a powder 10 be compres sed as described
aboye. As such, they are mea sures of the powder's ability 10 Powders
settle, and they permit an assessmenr of the relative (Ph. Eur. methad 2.9.45)
importance of inrerparticulate interactions. In a free-fiowing
powder, such interactions are less significant, and the bulk INTRODUCTION
and tapped densities will be closer in value. For more-poorly The wettability of solid surfaces is commonly characterised
fiowing materials, there are frequenrly greater inrerparticulate by direct or indirect conract angle measuremenrs.
inreractions, and a greater difference between the bulk and The conract angle (8) between a liquid and a solid is the
tapped densities will be observed. These differences are angle narurally formed when a drop of a liquid is placed on a
refiected in the compressibility index and the Hausner ratio . solid surface . This is depicted in Figure 2.9.45.-1. For a
2014 Appendix XVII T V -A519
given liquid, wettable solids show a low contact angle and

non-wettable solids show a contact angle of 90° or more. gas
'1s
salid
Figure 2.9.45.-1. - Contact angle (e) of a sessile drop observed

on a non-porous surface
2 methods for the determination of wettability are described

below. The methods are capable of measuring the wettability Figure 2.9..1-5.-2. - SessiLe drop determination wiLh visual
of porous solids like powders or granules. Both methods inJpectian qf the dropLet
express the wettability by a contact angle measurement
between the porous solid and a given liquido
PROCEDURE
The sessile drop method is based on direct measurement of a
contact angle of a sessile drop on a compacted powder disco Since powders are unable to form a completely flat surface,
the powder is usually compacted as a disc in an attempt to
With the Washburn method the contact angle is indirectly
make the surface smoother. A liquid drop with a given
measured. The method is based on the capillary effect of the
volume is placed on the disc (see Figure 2.9.45.-2) allowing
powder pores. The effect (mass gain) is recorded by special
direct measurement of the contact angle using a goniometer
electronic balances starting the moment when the powder
fitted with an eyepiece pro tractor, or by geometric
sample touches the surface of a liquid, preferably not
construction on a photomicrograph. Other physical and
dissolving or poorly dissolving the sample. The measurement
mathematical procedures of data analysis may also be
has very little or no effect on the state of the powder.
appropriate. The drop volume may influence the result.
Any pre-treatment of the sample to be examined is Several determinations of the contact angle (e) (n = 6) are
disadvantageous, since' the properties may be significantly usually carried out and the average is calculated.
altered. For example, the compaction of a powder as a disc
may de crease the surface free energy when the crystalline WASHBURN METHOD
state of the powder is changed (e.g. metastable forms), or The Washburn method is able to measure the contact angle
may increase surface free energy by creating crystal defects of porous solids with a contact angle in the range of 0-90 0
•
(disadvantage of the sessile drop method since compacted The tested material is the combination of the sample, the
powder discs are tested) . holder and the filter system. Therefore, an estimation or
The methods are usually applied to examine the following determination of the true value is not possible and only
parameters: apparent values of the contact angle can be determined.
- batch-to-batch consistency of samples in terms of However, the contact angle of the sample is the functional
wettability; property on which the result is significantly dependent.
- effect of liquid viscosity on wettability; The outcome of the test is a ranking order listing the
- effect of surface tension of a liquid on wettability; wettability of different substances or formulations
characterised by an apparent contact angle.
- alteration of surface properties of samples.
PRINCIPLE
SESSILE DROP METHOD If a porous solid is brought into contact with a liquid, such
This method may be used to characterise directly the that the solid is not submerged in the liquid, but rather is
wettability of coatings and compacted formulations such as just touching the liquid surface, then the rise of liquid into
tablets. Moreover, it is sometimes possible to use the sessile the pores of the solid due to capillary action will be governed
drop instrument in a dynamic measurement (dynamic by the following equations :
contact angle measurement, Figure 2.9.45.-2) of porous
solid/liquid systems where the contact angle decreases. 2 t
m = - (1)
By taking several contact angle measurements as a function A
of time, the rate of spreading accompanied by penetration of
a liquid droplet into a slightly porous solid may be studied. m mass of liquid sucked into the sol id;
Under equilibrium conditions the contact angle of a sessile time elapsed since the solid and the liquid were
drop depends on 3 intertelated surface tensions and is brought into contactó
determined using Young's equation (see Figure 2.9.45.-2, 1st A constant, dependent on the properties of the liquid
part): and the solid to be examined, calculated using the
following equation:
,s = ,SL +,L cose
A = r¡
Ys surface tension of the solid with airó e x p2 x , X cas e (2)
YSL interfacial tension of the solid with the liquid;
YL surface tension of the liquid with air. viscosity of the liquid;
V-A520 Appendix XVII T 2014
p density of the liquid; Table 2.9.45.'1. - Technical parameters ofthe electronic

y surface tension of me liquid; balance
8 contact angle between the solid and the Iiquid; Lift Mass measurement
e material constant, dependent on the porous texture of Range > 110 mm 0·210 g
the solido 10 ~g
Resolution O.llJm
Equations (1) and (2) lead to equation (3): Speed 0.099 . 500 mm/ min
m2 r¡
cos () = -t- x e x p2 x , (3)
A B
~---tJ
In setting up a Washburn determination, a Iiquid with known
density (p), viscosity (11), and surface tension (y) is used.
Under these conditions, when the mass of liquid rising into
the porous solid is monitored as a function of time (such that
capillary penetration rate «(~) is the experimental data), 2
unknowns remain according to equation (3): the contact
angle (8) of the liquid on the solid, and the solid material
constant (e) .
Determination ofthe material constant (e) The
material constant for a porous solid is determined by the
following equation, considering cylindrical pores:
(4)
E
r average capillary radius within the porous solid;
=
N = number of capillaries per volumetric unit.
If a Washburn determination is performed with a liquid
0
considered to have a contact angle of 0 (cos 0° = 1) on the -F
sol id, then the so lid material constant (e) is the only
remaining unknown in equation (3) and can thus be
determined. n-Heptane is the liquid of choice for determining
material constants because of its low surface tension (20.14 A. electronic balance C. sample holder E. immersion liquid
mN·m- 1 at 25 OC) . n-Hexane may also be used (18.43 B. computer D. filler F.lift
mN·m- 1 at 25 oC) but is more volatile. Ifthe powder
dissolves too quickly in these liquids, hexamethyldisiloxane Figure 2.9.45.-3. - Apparatus for contact angle
may be used instead (15.9 mN·m- 1 at 25 OC). Replicate measurement by the Washburn method
determinations are performed (n = 6) and the average value
calculated.
Sample holders The sample holder may be a small glass
Once the material constant (e) has been determined for the cylinder with a sintered-glass filter at one end.
solid to be examined, a sample of the solid can be tested for
Powder material holders (se e Figure 2.9.45-4) may also be
wettability by another liquidoThe material constant
made of aluminium; they are les s fragile than mose made of
determined by me n-heptane test is used in the Washburn
glass and have small holes in the bottom that render them
equation, in combination with the capillary penetration rate
easier to clean than a sintered-glass filter. The cover for the
«(~) data 'obtained while testing the substance to be
cell is equipped with 2 screw threads. One connects it with
examined in the prescribed liquido This allows calculation of
the sample chamber while the other allows the user to guide
the contact angle.
a piston down onto the sample itself and compact it.
NOTE: if a series of liquids (at least 2 liquids in addition to The apparatus is similar to an automatic tensiometer, except
the liquid used to determine the material constant) is tested for the sample holder.
against a given solid then the resultant contact angle data can
be used to calculate the surface energy of the porous solid.
APPARATUS
Figure 2.9.45.-3 shows me principal components ofthe
apparatus. The main device is an electronic balance with a
suitable processor ensurlng a suitable resolution in force
measurement and a suitable resolution in lifting up the
immersion Iiquid towards me sample.
Table 2.9.45.-1 indicates parameters ofthe electronic balance
that are generally considered suitable.
2014 Appendix XVII U V-A521
powder sample may also be mounted on the electronic

balance. A sample holder is omitted in this case.
Afrer connecting ro the balance, the sample holder is
positioned with the porous solid just aboye the surface of the
liquid (see Figure 2.9.45.-3), using the lifr.
The liquid is raised further until it just touches the bottom of
the porous sample. Mass-versus-time data is then collected as
liquid penetra tes into the solid. Data can be presented in
either graphical or tabular format. The apparatus may
perform the whole determination automatically.
CRITICAL PARAMETERS
The following points must be considered.
Sample properties:
- water content of the sample;
- crystalline or solid-state propenies of the sample
(polymorphic form, type of solvate).
Sample preparation:
- homogeneity of any powder blend ro be examined;
View 01 plunger botlom View 01 apparalus botlom - particle-size distribution; before testing it is sometimes
advisable to sieve the sample (e.g. using a 250 ¡.lm sieve);
A. fixing C. thread E. capillary holes
- the optimal compaction parameters (amount of sample,
B. cover D. plunger F. capillary holes number of taps or piston mass) must be determined;
Figure 2.9.45.-4. - Example of sample holder with plunger for - the compaction sta te of the different powder samples must
compaction of a powder be uniform;
- the sample holder or, if used, the glass frit must be
carefully cleaned;
PROCEDURE
- uniformity of the results is improved by using a sample
Filling of the sample holder holder made of aluminium.
Place a disc of filter paper in the bottom of the aluminium or Immersion liquid:
glass sample holder. This prevents powder from leaking out
- specifications of the irnmersion liquid must be indicated.
of the bottom of the cell. The filter do es not have to be made
of paper, but it must be a material that is easily wetted by the
liquid to be tested. A black-band filter (used for reverse
osmosis) is recommended because of its high porosity and
minimum flow resistance. U. Crystallinity
Place a known amount of powder into the cell. (Ph. Eur. method 5.16)
The reproducibility of material constants and contact angles This chapter provides general information on crystallinity and
will depend on the ability ro weigh out the same amount of refers to the various techniques described in the European
powder for each test when a sufficient and adjusted amount Pharmacopoeia that are used for its determination.
of powder is compacted in a uniform way INTRODUCTION - THE CONCEPT OF CRYSTALLlNITY
(i.e. tappinglcompaction of the powder). Most organic and inorganic compounds of pharmaceutical
For most powders, a correct amount is in the range of a few relevance exist as a solid material, which can be characterised
grams, typically filling about 2/3 of the capacity of the holder. by a structure located between a perfectly ordered crystal and
Place a second piece of filter paper on rop of the powder in an amorphous material.
the cell. This will prevent powder from rising through the Real crystals lie somewhere between an ideal crystal state and
holes in the piston dunng the compaction process and/or the amorphous state. The position of a crystal on a scale
during the determination. bounded by these 2 ex.tremes is termed its crystallinity.
Tapping/compaction of the powder A perfectly ordered crystal is an ideal state that is seldom, if
A bulk powder bed is very porous and thus very sensitive ro ever, achieved. The structural units of a crystal, termed unit
small influences that can easily alter the porosity and cells, are repeated regularly and indefinitely in 3 dimensions
consequently the c-constant. Therefore a tapped powder may in space. The unit cell has a definite orientation and shape
be advantageous and will show more reproducible results. defined by the translational vecrors a, b and e, and the angles
The appropriate number of taps must first be evaluated: CJ., Pand y, and hence has a definite volume, V, that contains
50-100 taps are usually appropriate. the atoms and molecules necessary for forming the crystal.
If the aluminiurn sample holder is used then it may be A crystalline system is defined by 3 long-range order
mounted in the cylinder of a stamp volumeter, which can run symmetry operarors (translational, orientational and
the evaluated number of taps. conformational); the various mesophases (liquid crystals,
If tapping is not appropriate, the powder bed is compacted crystals and plastic crystals) have 1 or 2 of the long-range
by screwing the piston of the aluminium sample holder symmetry operators and the ideal amorphous state is defined
applying a specified pressure. by the absence of all 3 operators.
A further possibility is centrifugation under defined Each crystal can be classified as a member of one of 7
conditions. Where applicable, a compacted disc of the possible crystal systems that are defined by the relationships
between the individual dimensions a, b and e and between
V-A522 Appendix XVII U 2014
the individual angles rt., ~ and y of the unit cel!. X-ray powder difIraction (2.9.33) XRPD is still the
The structure of a given crystal may be c1assified according most commonly used method for deterrnining the degree of
to one of the 7 crystal systems, to one of the 14 Bravais crystallinity, although this method suffers from sorne
lattices and to one of the 230 space groups. All the 230 limitations due to peak broadening, amorphous halo and
possible space groups, their symmetries and the symmetries preferred oóentation, which make interpretation and
of their diffraction patterns are compiled in the International quantitation difficult.
Tables for Crystallography. XRPD alone is often insufficient to distinguish between the
Many substances are capable of crystallising in more than different non-crystalline phases. The X-ray diffraction pattem
one type of crystal lattice, which is known as polymorphism. of a purely amorphous and nanocrystalline phase is
The occurrence of polymorphism is a common phenomenon characteristic of a broad diffuse halo. In-depth analysis of the
among organic molecules, giving rise to different physico- X-ray diffraction patterns will show that the diffuse halo in
chemical properties. Crystalline polymorphs have the same the pattem of nanocrystalline material shows sorne
chemical composition but different internal crystal structures correlation to the pattem of the parent crystalline phase,
and, therefore, possess different physico-chemical properties. while in the case of apure amorphous phase such a
The different crystal structures in polymorphs are due to correlation does not existo Additional techniques may be
different atomic packing arrangements andlor different required to establish the tme nature of X-ray amorphous
conforrnations of the molecules (see chapter 5.9. materials.
Polymorphism) . Thermal analysis Therrnal analysis (2.2.34) of crystalline
The other extreme of a crystal state is the ideal or tme materials exhibits a melting transition that is often
amorphous state, where alllong-range order is 10s1. For most accompanied by decomposition or evaporation of solvents.
organic systems certain short-range order remains, but this is In the case of tme amorphous materials, thermal analysis
not expected to extend over distances much larger than reveals a glass transition, whereas only a melt would be
nearest neighbour (NN) or next nearest neighbour (NNN) expected for a nanocrystalline materia!.
interactions, which are typically lessthan 2-2.5 nm for small Microcalorimetry (2.2.61) It is a highly sensitive
organic molecules. technique which allows the determination of the rate and
Amorphous mateóal is characteósed by the absence of extent of chemical reactions, changes of phase or changes of
distinct reflections in the X-ray powder diffraction (XRPD) structure. Amorphous parts of a substance can recrystallise
pattern (2.9.33). by subjecting the sample to higher relative humidity or an
The crystallinity of a real powder can be considered by 2 atrnosphere containing organic vapour. The measurement of
models of crystallinity. In the l-state model all partic1es will the heat of recrystallisation enables the amorphous content to
be of the same crystallinity whereas in the 2-state model each be deterrnined from the enthalpy of recrystallisation.
partic1e can be either crystalline or amorphous, such that the By relating the output from the micro calo rime ter for a
actual crystallinity of the powder is the weighted average of sample to that obtained for an amorphous standard, it is
these 2 extreme crystallinities. Such a powder is obtained possible to quantify the amorphous content of the sample.
when pure crystalline and amorphous phases are physically The range of amorphous content covered by this method
mixed. In reality, a powder probably contains partic1es with depends on the individual substance to be tested;
different degrees of crystallinity, just as it may contain in favourable cases limits of detection below 1 per cent can
partic1es with different sizes and shapes. be reached .
The extent of disorder in a crystalline solid can affect many Solution calorimetry (2.2.61) Solution calorimetry
physico-chemical properties of substances for pharrnaceutical provides a means of determining enthalpy of solution for a
use. Because of the great relevance of these properties, it is solid substance. The crystallinity of the solid sample to be
important to be able to assess the extent of disorder or the examined is given by the enthalpy of solution of the solid
crystallinity of a solid by a suitable quantitative method. sample LiHx' minus the enthalpy of solutio? of th~ chosen
reference standard of the same substance LiH: when
METHODS FOR MONITORING ANO determined under the same conditions. Because the reference
DETERMINING CRYSTALLINITY standard is usually chosen for its perceived high crystallinity,
Vaóous methods are .available for deterrnining the its enthalpy of solution is usually algebraically greater (more
crystallinity of a solid. Many techniques cannot detect or endotherrnic or less exothermic) than that of the solid
quantify these properties independently; for this reason, it is sample to be examined in the same solvento Consequently,
useful to combine several of the methods descóbed below. the crystallinity determined is a negative quantity with the SI
Such methods often do not give accurate results and limits of units kJ/mol or J/g O/kg is avoided because of its
quantitation are usually much greater than those for chemical unwieldiness and potential for error). The preference for a
impurities. In addition, certain assumptions have to be made negative value with respect to a highly crystalline reference
about the relationship between standards used for calibration, standard recognises the fact that most samples have a lower
which are typically mixtures of crystalline and amorphous crystallinity than this reference standard.
partic1es (2-state model), and the samples to be analysed that Near-infrared (NIR) spectrophotometry Near-infrared
are likely to have at least a small component of mateó al (NIR) spectrophotometry (2.2.40) is another technique used
exhibiting l-state model behaviour. Finally, the lack of well- to measure the degree of crystallinity, and has also been
defined standards for 100 per cent crystalline or 100 per cent proven to be useful in studies of polymorphism. The NIR
amorphous mateóal complicates the validation of such spectmm of a sample contains both physical and chemical
methods. As explained aboye, it is obvious that different information. Being non-invasive, non-destmctive and
amorphous or non-crystalline phases exist and even co-exist operable at room temperature, the method is a valuable tool
in a solid powder. These different non-crystalline forrns of a to assess changes in the amorphous and crystalline state.
solid can give different responses depending on the Infrared absorption spectrophotometry and Raman
techniques used for deterrnining the degree of crystallinity. spectrometry Infrared absorption spectrophotometry
2014 Appendix XVII V V-A523
(2.2.24) and Raman spectrometry (2.2.48) are other great importance during the development and subsequent
techniques used to measure the degree of crystallinity, and manufacture of a pharmaceutical preparation.
have also been proven to be useful in studies of In reality, a powder probably contains partic1es with different
polymorphism. The IR spectrum and Raman spectrum of a degrees of crystallinity, just as ir may contain particles with
sample contain both physical and chemical information. varying sizes and shapes. The lower the crystallinity of a
Solid-state NMR Solid-state nuclear magnetic resonance solid, the greater its enthalpy and entropy. The increase in
spectrometry (ss NMR) (2.2.33) can be used 10 provide enthalpy is never 10tally compensated for by the increase in
information about polymorphism and related relative entropy; therefore, the Gibbs free energy, which reflects the
molecular conformations. However, sorne caution has to be balance between them, actually increases. Hence, the lower
exercised in the interpretation of results, since it is not the crystallinity of a material (powder), and consequently the
always simple to distinguish between samples that comprise a greater its amorphous character, the greater its apparent
mixture of different physical forms (2-state model) and those intrinsic solubility and dissolution rate, but the lower its
that comprise crystals having disorder with exchange that is thermodynamic stability. Because of the great relevance of
slow on the NMR timescale. Similarly, samples that contain these properties, crystallinity is also an important property
defects arising from different molecular conformations or and requires measurement by a suitable method.
slightly different packing arrangements (l-state model) may In the following chapter, the crystallinity or the content of
show additional signals in the spectra. Solid-state NMR may amorphous parts of a powder are measured by calorimetric
be quite sensitive 10 this, even if lattice parameters are hardly methods such as microcalorimetry or solution calorimetry,
affected and, consequently, little or no change is observed by although other methods could be used (e.g. see general
XRPD. It is evident that the crystallinity of substances for chapter 2. 9.33. Characterisation oi crystalline and partially
pharmaceutical use can be complex, and both crystalline crystalline solids by X-ray powder diffraction (XRPD) ) .
defects and amorphous material may co-exist.
Many substances are capable of crystallising in more than
Optical microscopy A method 10 detect whether or not one type of crystallattice, which is known as polymorphism.
particles are crystalline is 10 use a polarising microscope If water or a solvent is incorporated in the crystallattice the
(2.9.37), where particles show birefringence and extinction crystals are termed hydrates or solvates. Because of the
positions when the microscope stage is revolved. different crystal packing, and/or molecular conformation and
lattice energy, they usually exhibit different physical
properties. For simplicity, calorimetry measurements for
degree of crystallinity determination discussed here assume
V. Characterisation of Crystalline Solids only one solid crystalline form present in the material of
interest. The theory and experimental technique can be easily
by Microcalorimetry and Solution expanded to polymorphic systems with proper consideration
Calorimetry of the enthalpy differences among the polymorphs.
METHOD 1 - MICROCALORIMETRY
For the purpose oi this chapter, crystalline material, partially
(DETERMINATION OF AMORPHOUS
crystalline material and amorphous material are considered as
CONTENT)
solzds.
Most chemical, physical and biological processes are
INTRODUCTION - THE CONCEPT OF associated with the exchange of heat. Microcalorimetry is a
CRYSTALLINITY highly sensitive technique 10 monitor and quantify both
exothermic (heat producing) and endothermic (heat
The perfectly ordered crystallattice with every molecule in its
absorbing) changes associated with those processes.
expected lattice position is an ideal that is seldom, if ever,
The technique allows the determination of the rate and
achieved. The other extreme is the amorphous sta te, in
extent of chemical reactions, changes of phase or changes of
which a solid c'ontains the maximum possible density of
imperfections (defects of various dimensionalities), such that structure.
alllong-range order is lost while only the short-range order, Thermal events prodticing only a fraction of a microwatt can
imposed by its nearest neighbours, remains. Real crystals lie be observed using microcalorimetry. This means that
somewhere between these 2 extremes. A crystal's position on temperature differences less than 10-6 K must be detectable .
a scale bounded by these 2 extremes is termed crystallinity. Microcalorimetry typically uses the heat flow (heat leakage)
principie, where the heat produced (or absorbed) in a
All real crystals, even in the pure sta te, possess sorne lattice
thermally defined vessel flows away (or into) in an effort to
imperfections or defects, which increase both the energy
re-establish thermal equilibrium with its surroundings.
(enthalpy under conditions of constant atmospheric pressure)
Exceptional thermal stability with its surrounding has to be
and the disorder (expressed as the entropy) of the crystal
achieved either by a heat sink or an electronically regulated
lattice. A crystal with a relatively low density of imperfections
surrounding.
is said to be highly crystalline and to possess a high
crystallinity. By contrast, a particle with a relatively high Heat energy from an active sample in the reaction vessel is
density of imperfections is said 10 be partially amorphous and channelled typically through Peltier elements; they act as
10 possess a low crystallinity. In ideal terms, a totally thermoelectric generators using the Seebeck effect. The heat
amorphous particle corresponds 10 zero crystallinity. energy is converted into a voltage signal proportional 10 the
Amorphous particles may contain somewhat ordered heat flow.
domains that can act as nuclei for crystallisation; such Results are typically presented as a measure of the thermal
so-called amorphous particles are said to possess a low-Ievel energy produced per unit of time (Watt) as a function of
but finite crystallinity. time.
The ability 10 detect and to quantify the amount of
amorphous material within a highly crystalline substance is of
V-A524 Appendix XVII V 2014
APPARATUS taking place simultaneously, such as the absorption of water

Microcalorimeters are typically designed as twin systems with vapour into the amorphous parts of the powder and the
a measuring vessel and a reference vessel. Vessels are generation of water vapour from the test-tube. After this
typically made of glass or stainless steel. For certain initial response there is a large exothermic response caused
applications specially designed ves seIs which allow the by the recrystallisation of the amorphous material. Also
addition of a gas, a liquid or a solid material may be used. inc1uded, but not seen, are the expulsion of excess water
from the recrystallised parts and its condensation. Thus, the
CALIBRATION area under this exothermic recrystallisation response is
The microcalorimeter is calibrated for heat fiow (energy per proportional to the heat of recrystallisation.
time unit) using either calibrated external or internal
electrical heat sources or a suitable standard reaction. 2.5
SENSITIVITY
JI
The sensitivity of the microcalorimetric method can be
assessed based on an appropriate standard sample analysed 2
according to the corresponding method in conjunction with
the determination of the instrument baseline noise.
PROCEDURE ~ 1.5
:s
Weigh in a suitable vessel an appropriate quantity of the
substance to be examined. Close the vessel carefully to avoid
any evaporation of solvents and place the vessel in the sample 1
holder. If appropriate, allow the vessel to equilibrate at the
temperature of the measurement before placing it in the
measuring position.
0.5
Begin the analysis and record the heat fiow, with the time on
the abscissa and the heat fiow on the ordinate (specify the
direction of exothermic or endothermic heat fiow).
DETECTION AND QUANTIFICATION OF
o~======~-----L~~===-
AMORPHOUS CONTENT IN POWDERS o 1 2 3
The amorphous state is metastable with respect to the Time ( Hours )
crystalline state; recrystallisation may therefore occur.
The measurement of the heat of recrystallisation enables the Figure 2.2.61.-1. - Typical microcalorimetric output of power
amorphous content to be determined by the area of the (in IJ W) as a function of time (in hours): amorphous collapse
recrystallisation peak. By relating the output from the peak (1) and crystallisation peak (11) for mainly amorphous
microcalorimeter for a sample to that obtained from an lactose at 25 oc and 75 per cent relative humidity
amorphous standard, it is possible to quantify the amorphous
content of the sample. The range of amorphous content
covered by this method depends on the individual substance METHOD 2 - SOLUTION CALORIMETRY
to be tested; in favourable cases limits of detection below
(DETERMINATION OF CRYSTALLINITY)
1 per cent can be reached. Solution calorimetry provides a means of determining
Recrystallisation can be initiated by subjecting the sample to enthalpy of solution (i.e. heat of solution under constant
higher relative humidity or an atrnosphere containing organic atrnospheric pressure) of a substance. Enthalpy of solution is
vapour. The sample is typically placed in an ampoule which defined as the enthalpy of the substance dissolved in the
also contains a small test-tube containing an aqueous solution to a defined concentration minus the enthalpy of the
saturated salt solution, an organic solvent, or a solvent original substance. Thé solvent for the dissolution process
mixture. must be such that the mas s of solid dissolves within a time
frame that matches the response time of the calorimeter, as
The heat of recrystallisation is typically measured using a
discussed below. The enthalpy of solution is proportional to
fixed sample mass placed in a glass or steel vessel. The test-
the amount of solid being dissolved. This amount may be
tube containing a saturated salt solution or an organic solvent
defined as 1 mol for molar enthalpy or as 1 g for specific
is chosen to be large enough to allow a full saturation of the
enthalpy. If the substance possesses adequate purity (as
atrnosphere aboye the sample. The mass of the sample and
determined by the degree of accuracy required) and if its
the nature of the vapour atrnosphere aboye the sample are
molecular mass is known, the molar enthalpy is preferred,
chosen so that recrystallisation occurs in such a way that a
otherwise the specific enthalpy must be used. The enthalpy
distinct peak is observed, c1early separated from initial
of solution is weakly dependent on both the temperature,
thermal events caused by introduction of the sample.
which is usually 25.0 oC, and the final concentration of the
The conditions under which the transition of the amorphous dissolved solute.
phase to a rhermodynamically more stable crystalline state
It is usually preferred to express the crystallinity, P" of a
occurs will have a significant impact on the time of
substance on a percentage scale . This procedure requires 2
recrystallisation. In particular, physical mixtures of purely
reference standards, namely a highly crystalline sample
amorphous and crystalline material will behave differently
assuming 100 per cent crystallinity and having a measured
from a partially crystalline material. These effects should be
enthalpy of solution of ~Hcs, and an amorphous sample
considered when developing a method.
assuming O per cent crystallinity and having a measured
A typical response for the recrystallisation of a mainly enthalpy of solution of ~H:. From these values and from the
amorphous material is shown in Figure 2.2.61.-1. The first measured enthalpy of solution, ~H,', of the solid under
part of the curve represents several concurrent processes
2014 Appendix XVII V V-A525
study, the percentage crystallinity of the solid, Pe> may be compensation process elimina tes the effects of heat losses to
ca1culated as follows : or heat gains from the bath. Therefore, isothermal solution
calorimeters are more advantageous than isoperibol solution
Pe (%) = 100 (6.H: - 6.H~) / ( 6.H~ - 6.H~) calorimeters when the solution process is relatively slow.
SOLUTION CALOR/METER CALIBRATION
Clearly, crystallinity expressed on a percentage scale depends
To ensure the accuracy of the calorimeter, chemical
on 3 measured values and the enthalpies of solution may be calibrations must be performed on a regular basis. For an
replaced by other corresponding physical quantities that endothermic solution process, the calibration of the
depend on crystallinity. The value of the percentage calorimeter is checked by measuring the heat absorbed
crystallinity of a sample, however, depends not only on the during the dissolution of potassium chloride in distilled water
nature and method of preparation of the 2 reference at 298.15 K (25.0 oC). The established enthalpy change in
standards, but also on the choice of the physical quantity that this endothermic process is 235.5 J/g (17.56 kJ/mol) . For an
is measured. exothermic solution process, the calorimeter is checked by
The enthalpy of solution is measured either by an isoperibol measuring the heat evolved during the dissolution of 5 g per
(constant perimeter, i.e. jacket) solution calorimeter or by an litre of tromethamine [tris(hydroxymethyl)aminomethane,
isothermal (constant temperature) solution calorimeter. THAM) in a 0.1 mol/L aqueous hydrochloric acid solution
Typically, at least 3 measurements are made with each at 298.15 K (25.0 oC) . The established heat for the
sample. The mean of these values is then ca1culated. aforementioned process is -246 .0 J/g (-29 .80 kJ/mol).
The exact requirements will depend upon the equipment
capability and degree of accuracy needed. SAMPLE HANDLING
The chemical and physical stability of solids may decrease
ISOPERIBOL SOLUTION CALORIMETRY with decreasing crystallinity. In particular, solids of low
In the isoperibol solution calorimeter, the heat change during crystallinity, especially amorphous solids, tend to sorb water
the solution process causes a corresponding change in vapour from the atrnosphere, leading to crystallisation and a
temperature of the solvent-solute system (i.e. solution) . This corresponding gain in crystallinity. For these reasons,
temperature change is measured by a temperature sensor, anhydrous samples whose crystallinity is to be deterrnined
which is wired to an electrical circuit that records an must be sto red at zero humidity or below critical humidity
electrical signal corresponding to the temperature change. levels in sealed chambers containing a desiccant, preferably
Typically, this temperature change in an electronic form is containing an indicator of effectiveness. If crystallinity-
measured at precisely defined time intervals to produce humidity studies are to be carried out, the sample is stored in
temperature-time data that are collected, analysed by a a sealed chamber containing a saturated salt solution to
computer, and then plotted. A blank run without addition of provide a defined relative humidity.
the solid solute to the solvent norrnally shows no discernible
change in the slope of the temperature-time plot.
For isoperibol solution calorimeters, response is fairly rapid,
but corrections must be made for any heat losses to or heat
gains from the bath. Therefore, isoperibol solution
calorimeters are more advantageous than isotherrnal solution
calorimeters when the solution process is relatively fast.
For all measurements of enthalpy of solution using isoperibol
solution calorimeters, the choice of solvent is critica!.
The nature and mass of the solvent and the mas s of sample
alIow the total heat change, corresponding to total dissolution
of the solid, to proceed to completion within 5 min under
vigorous stirring at a constant rotational speed within the
range of 400-600 r/min.
The effective heat ·capacity of the calorimeter cell and its
contents is deterrnined for every calorimeter runoThis
deterrnination is accomplished by electrical heating of the
contents of the calorimeter ceIl. The effective heat capacity is
deterrnined according to 1 of 2 protocols: either by making 1
deterrnination after ampoule breakage or by making 1
deterrnination before and a 2nd deterrnination after ampoule
breakage and then averaging the 2 results. The accuracy and
reliability of the electrical heating are established by the
accuracy and reliability of the aforementioned chemical
calibrations.
ISOTHERMAL SOLUTION CALORIMETRY
In the isothermal (constant temperature) solution
calorimeter, the heat change during the solution process is
compensated for by an equal but opposite energy change,
such that the temperature of the solvent-solute system
(i.e. solution) remains essentially constant. This equal but
opposite energy change is measured and, when its sign is
reversed, provides the enthalpy of solution. For isotherrnal
calorimeters, response is relatively slow, but the
V-A526 Appendix XVIII 2014
are required to maintain the sterility of the product

Appendix XVIII throughout its shelf-life.
Sterility Assurance Level (SAL)
Methods of Sterilisation Where appropriate reference is made within the methods
described below, to a "sterility assurance level" or "SAL".
(Ph. Eur. general texts 5.1.1,5.1.2 and 5.1.5)
The achievement of sterility within any one item in a
popularion of items submitted to a sterilisation process
Methods of preparation of sterile products
cannot be guaranteed nor can it be demonstrated.
(Ph. Eur. method 5.1.1) The inactivation of micro-organisms by physical or chemical
Sterility is the absence of viable micro-organisms. means fol!ows an exponential law; thus there is always a
The sterility of a product cannot be guaranteed by testing; finite srarisrical probability that a micro-organism may survive
it has to be assured by the application of a suitably validated the sterilising process. For a given process, the probability of
production process. Ir is essential thar the effect of the survival is determined by rhe number, types and resisrance of
chosen sterilisation procedure on the product (including its the micro-organisms present and by the environment in
final container or package) is investigated to ensure which the organisms exisr during treatment. The SAL of a
effectiveness and the integrity of the product and that the sterilising process is the degree of assurance with which the
procedure is validated before being applied in practice. It is process in question renders a population of items sterile.
recommended that the choice of the container is such as to The SAL for a given process is expressed as the probability
al!ow the optimum sterilisation ro be applied. Failure ro of a non-sterile item in thar population. An SAL of 10- 6, for
follow mericulously a validated process involves the risk of a example, denotes a probability of nor more than one viable
non-sterile product or of a deteriorated product. Revalidation micro-organism in 1 x 10 6 sterilised irems of the final
is carried out whenever major changes in the sterilisation product. The SAL of a process for a given product is
procedure, including changes in the load, take place. It is established by appropriate validation srudies.
expecred rhat the principIes of good manufacruring pracrice
(as described in, for example, the European Community Methods and conditions of sterilisation
Guide to GMP) will have been observed in the design of the
Srerilisation may be carried out by one of the methods
process including, in particular, the use of:
described below. Modifications to, or combinations of, these
- qualified personnel with appropriate training, methods may be used provided that the chosen procedure is
- adequate premises, valida red both with respect to its effectiveness and the
- suitable producrion equipment, designed for easy cleaning integriry of the product including its container or package.
and sterilisation, For al! methods of sterilisation the critical conditions of the
- adequate precautions ro minimise the bioburden prior to operation are monitored in order to confirm that the
sterilisation, previously determined required conditions are achieved
- validated procedures for al! critical production steps, throughout the batch during the whole srerilisation process.
This applies in al! cases including those where the reference
- environmental monitoring and in-process testing
conditions are used.
procedures.
The precautions necessary to minimise the pre-sterilisation Terminal sterilisation
bioburden include the use of components with an acceptable For terminal sterilisation it is essential to take into account
low degree of microbial contamination. Microbiological the non-uniformity of the physical and, where relevant,
monitoring and setting of suitable action limits may be chemical conditions within the sterilising chamber.
advisable for ingredients which are liable to be contamina red The location within the sterilising chamber that is lea sr
because of their origin, nature or method of preparation. accessible to the sterilising agent is derermined for each
The methods described here appiy mainly to the inactivation 01' loading configuration of each type and size of container or
removal of bacteria, yeasts and moulds. For biological products of package (for example, the coolesr locarion in an autoclave).
animal or human origin or in cases where such material has been The mínimum lethality delivered by the sterilising cycle and
used in the production process, it is necessary during validation to the reproducibility of the cycle are also derermined in order
demonstrate that the process is capable of the removal or to ensure !hat al! loads will consistently receive the specified
inactivation of relevant viral contamination. Guidance on this treatment.
aspect is provided in, for example, the appropriate European Having established a terminal sterilisation process, knowledge
Community Notes for Guidance. of its performance in rourine use is gained wherever possible,
Wherever possible, a process in which the product is by monitoring and suitably recording rhe physical and, where
sterilised in its final container (terminal srerilisation) is relevant, chemical conditions achieved within the load in the
chosen. When a fully validated terminal sterilisation method chamber throughout each sterilising cycle.
by sream, dry heat or ionising radiation is used, parametric Steam sterilisation (Heating in an autoclave)
releas e, that is the releas e of a batch of sterilised items based Sterilisation by saturated steam under pressure is preferred,
on process data rather than on the basis of submitting a wherever applicable, especial!y for aqueous preparations.
sample of rhe items to sterility testing, may be carried out, For this method of terminal sterilisation the reference
subject to the approval of the comperent authority. conditions for aqueous preparations are heating at a
If terminal sterilisation is not possible, filtration through a minimum of 121 oC for 15 mino Other combinations of time
bacteria-retentative filter or aseptic processing is used; and temperature may be used provided that it has been
wherever possible, appropriate additional treatment of the satisfactorily demonstrated that the process chosen delivers
product (for example, hearing of the product) in its final an adequate and reproducible leve! of lethality when
container is applied. In al! cases, the container and closure operating routinely within the established tolerances.
The procedures and precautions employed are such, as to
give an SAL of 10- 6 or better. Guidance conceming
2014 Appendix XVIII V-A527
validation by means of the Fo concept is provided below penetration by gas and moisture imo the material to be
(5.1.5) . sterilised is ensured and that it is followed by a process of
Knowledge of the physical conditions (temperature and elimination of the gas under conditions that have been
pressure) within the autoclave chamber during the previously established to ensure that any residue of gas or its
sterilisation procedure is obtained. The temperature is usually transformation products in the sterilised product is below the
measured by means of temperature-sensing elements inserted concentration that could give rise to toxic effects during use
into representative containers together with additional of the producto Guidance on this aspect with respect to the
elements at the previously established coolest part of the use of ethylene oxide is provided, for example, in the
loaded chamber. The conditions throughout each cycle are appropriate European Community Notes for Guidance.
suitably recorded, for example, as a temperature-time chart, Wherever possible, the gas concentration, relative humidity,
or by any other suitable means. temperature and duration of the process are measured and
Where a biological assessment is carried out, this is obtained recorded. Measurements are made where sterilisation
using a suitable biological indicator (5.1.2). conditions are least likely to be achieved, as determined at
Dry heat sterilisation For this method of terminal validation.
sterilisation the reference conditions are a minimum of The effectiveness of the process applied to each sterilisation
160 oC for at least 2 h. Other combinations of time and load is checked using a suitable biological indicator (5. 1.2) .
temperature may be used provided that it has been A suitable sample of each batch is tested for sterility (2.6.1)
satisfactorily demonstrated that the process chosen delivers before the batch is released.
an adequate and reproducible level of lethality when
Filtration
operated routinely within the established tolerances.
The procedures and precautions employed are such as to Certain active ingredients and products that cannot be
give an SAL of 10- 6 or better. terminally sterilised may be subjected to a filtration
procedure using a filter of a type that has been demonstrated
Dry heat sterilisation is carried out in an oven equipped with to be satisfactory by means of a microbial challenge test using
forced air circulation or other equipment specially designed a suitable test micro-organismoA suspension of Pseudomonas
for the purpose. The steriliser is loaded in such a way that a diminuta (ATCC 19146, NCIMB 11091 or CIP 103020)
uniform temperature is achieved throughout the load. may be suitable. It is recommended that a challenge of at
Knowledge of the temperature within the steriliser during the least 10 7 CPU per cm 2 of active filter surface is used and
sterilisation procedure is usually obtained by means of that the suspension is prepared in tryptone soya broth which,
temperature-sensing elements inserted into representative after passage throug the filter, is collected aseptically and
containers together with additional elements at the previously incubated aerobically at 32 oC. Such products need special
established coolest part of the loaded steriliser. precautions . The production process and environment are
The temperature throughout each cycle is suitably recorded. designed to minimise microbial contamination and are
Where a biological assessment is carried out, this is obtained regularJy subjected to appropriate monitoring procedures.
using a suitable biological indicator (5.1.2) . The equipment, containers and closures and, wherever
Dry heat at temperatures greater than 220 oC is frequently possible, the ingredients are subjected to an appropriate
used for sterilisation and depyrogenation of glassware. In this sterilisation process . It is recommended that the filtration
case demonstratión of a 3-log reduction in heat resistant process is carried out as close as possible to the filling point.
endotoxin can be used as a replacement for biological The operations following filtration are carried out under
indicators (5.1.2) . aseptic conditions .
Ionising radiation sterilisation Sterilisation by this Solutions are passed through a bacteria-retentive membrane
method is achieved by exposure of the product to ionising with a nominal pore size of 0.22 ~lm or les s or any other type
radiation in the form of gamma radiation from a suitable of filter known to have equivalent properties of bacteria
radioisotopic source (such as cobalt 60) or of a beam of retention. Appropriate mea sures are taken to avoid loss of
electrons energised by a suitable electron accelerator. solute by adsorption on to the filter and to avoid the release
In some countries there are regulations that lay down rules for the of contaminants from the filter. Attention is given to the
use of ionising radiation for sterilisation purposes, for example, in bioburden prior to filtration, filter capacity, batch size and
the appropriate European Community Notes for Guidance. duration of filtration. The filter is not used for a longer
period than has been approved by validation of the
For this method of terminal sterilisation the reference
combination of the filter and the product in question.
absorbed dose is 25 kGy. Other doses may be used provided
that it has satisfactorily been demonstrated that the dose The integrity of an assembled sterilising filter is verified
chosen delivers an adequate and reproducible level of before use and confirmed after use by carrying out tests
lethality when the process is operated routinely within the appropriate to the type of filter used and the stage of testing,
established tolerances. The procedures and precautions for example bubble-point, pressure hold or diffusion rate
employed are such as to give an SAL of 10- 6 or better. tests.
During the sterilisation procedure the radiation absorbed by Due to the potential additional risks of the filtration method
the product is monitored regularly by means of established as compared with other sterilisation processes, a prefiltration
dosimetry procedures that are independent of dose rateo through a bacteria-retentative filter may be advisable in cases
Dosirneters are calibrated against a standard source at a where a low bioburden cannot be ensured by other means .
reference radiation plant on receipt from the supplier and at Aseptic preparation
suitable intervals of not longer than one year thereafter. The objective of aseptic processing is to maintain the sterility
Where a biological assessment is carried out, this is obtained of a product that is assembled from components, each of
using a suitable biological indicator (5.1.2). which has been sterilised by one of the above methods. This
Gas sterilisation This method of sterilisation is only to be is achieved by using conditions and facilities designed to
used where there is no suitable alternative . It is essential that prevent microbial contamination. Aseptic processing may
V-A528 Appendix XVIII 2014
include aseptic filling of products into container/closure a) the resistance of the test strain is suitable for the
systems, aseptic blending of formulations followed by aseptic particular sterilisation method and is great compared to
filling and aseptic packaging. the resistance of micro-organisms potentially
In order to maintain the sterility of the components and the contaminating me product;
product during processing, careful attention needs to be b) the test strain is non-pathogenic;
given to: c) the test strain is easy to culture.
- environment, After incubation, growth of the reference micro-organisms
- perso=el, subjected to a sterilisation procedure indica tes mat the
- critical surfaces, procedure has been unsatisfactory.
- container/closure sterilisation and transfer procedures, Steam sterilisation The use of biologicaI indicators
- maximum holding period of the product before filling into intended for steam sterilisation is recommended for the
the final container. validation of sterilisation cycles. Spores of Geobacillus
stearothermophilus (for example, ATCC 7953, NCTC 10007,
Process validation includes appropriate checks on all the NCIMB 8157 or CIP 52.81) or other strains ofmicro-
aboye and checks on the process are regularly carried out by organisms having demonstrated equivalent performance are
means of process simulation tests using microbial growth recommended. The number of viable spores exceeds 5 x
media which are then incubated and examined for microbial 10 5 per carrier. The D-value at 121 °C is not less than
contamination (media fill tests). In addition, a suitable 1.5 mino It is verified that exposing the biological indicators
sample of each batch of any product that is sterilised by to steam at 121 ± 1 oC for 6 min leaves revivable spores,
filtration andlor aseptically processed is tested for sterility and that mere is no growth of me reference micro-organisms
(2.6.1) before the batch is released. after me biological indicators have been exposed to steam at
121 ± 1 oC for 15 mino
Biological indicators of sterilisation
Dry heat sterilisation Spores of Bacillus atrophaeus (for
example, ATCC 9372, NCIMB 8058 or CIP 77.18) or
Biological indicators are standardised preparations of selected omer strains of micro-organisms having demonstrated
micro-organisms used to assess me effectiveness of a
equivalent performance are recommended for the preparation
sterilisation procedure. They usually consist of a population
of biological indicators. The number of viable spores exceeds
of bacterial spores placed on a suitable inert carrier. 1 x 10 6 per carrier and me D-value at 160 oC is not less
The inoculated carrier is covered in such a way mat it is
man 2.5 mino Dry heat at temperatures greater than 220 oC
protected from any deterioration or contamination, while
is frequently used for sterilisation and depyrogenation of
allowing me sterilising agent to enter into contact wim the
glassware. In mis case, demonstration of a 3 log reduction in
micro-organisms. Spore suspensions may be presented in heat-resistant bacterial endotoxin can be used as a
sealed ampoules. Biological indicators are prepared in such a
replacement for biological indicators.
way that they can be stored under defined conditions;
an expiry date is seto Ionising radiation sterilisation Biological indicators may
be used to monitor routine operations, as an additional
Micro-organisms of the same bacterial species as the bacteria
possibility to assess the effectiveness of me set dos e of
used to manufacture me biological indicators may be
radiation energy, especially in me case of accelerated electron
inoculated directly into a liquid product to be sterilised or
sterilisation. The spores of Bacillus pumilus (for example,
into a Iiquid product similar to mat to be sterilised. In mis
ATCC 27142, NCTC 10327, NCIMB 10692 or CIP
case, it must be demonstrated mat the liquid product has no
77.25) or omer strains of micro-organisms having
inhibiting effect on the spores used, especially as regards meir
demonstrated equivalent performance are recommended.
germination.
The number of viable spores exceeds 1 x 107 per carrier.
A biological indicator is characterised by the name of me The D-value is not less than 1.9 kGy. It is verified that mere
species of bacterium used as me reference micro-organism, is no growth of the reference micro-organisms after me
the number of me strain in me original collection, me biological indicators have been exposed to 25 kGy (minimum
number of viable spores per carrier and me D-value. absorbed dose) .
. The D-value is the value of a parameter of sterilisation
Gas sterilisation The use of biological indicators is
(duration or absorbed dose) required to reduce the number
necessary for all gas sterilisation procedures, both for the
of viable organisms to 10 per cent of me original number.
validation of me cycles and for routine operations.
It is of significance only under precisely defined experimental
Gas sterilisation is widely used for medical devices, isolators,
conditions. Only me stated micro-organisms are presento
chambers, etc. Use for such purposes is outside the scope of
Biological indicators consisting of more than one species of
me European Pharmacopoeia. The use of spores of Bacillus
bacteria on me same carrier may be used. Information on the
atrophaeus (for example, ATCC 9372, NCIMB 8058 or CIP
culture medium and the incubation conditions is supplied.
77 .18) or other strains of micro-organisms having
It is recommended that me indicator organisms are placed at demonstrated equivalent performance is recommended for
the locations presumed, or wherever possible, found by emylene oxide. The number of viable spores exceeds 1 x
previous physical measurement to be least accessible to me 10 6 per carrier. The parameters of resistance are the
sterilising agent. After exposure to the sterilising agent, following: the D-value is not less than 2.5 min for a test
aseptic technique is used to transfer carriers of spores to the cycle involving 600 mg/L of ethylene oxide, at 54 oC and at
culture media, so mat no contamination is present at me 60 per cent relative humidity. It is verified that mere is no
time of examination. Biological indicators mat include an growth of the reference micro-organisms after me biological
ampoule of culture medium placed directly in me packaging indicators have been exposed to me test cycle described
protecting me inoculated carrier may be used. aboye for 25 min and mat exposing the indicators to a
A choice of indicator organisms is made such that: reduced temperature cycle (600 mg/L, 30 oC and
2014 Appendix XVIII V-A529
60 per cent relative humidity) for 50 min leaves revivable

spores.
Application of the Fo concept to steam sterilisation
of aqueous preparations
The following chapter is published for information.
The Fo value of a saturated steam sterilisation process is the
lethality expressed in terms of the equivalent time in minutes
at a temperature of 121 oC delivered by the process to the
product in its final container with reference to micro-
organisms possessing a theoretical Z-value of 10.
The total Fo of a process takes account of the heating up and
cooling down phases of the cycle and can be calculated by
integration of lethal rates with respect to time at discrete
temperature intervals.
When a steam sterilisation cycle is chosen on the basis of the
Fo concept, great care must be taken to ensure that an
adequate assurance of sterility is consistently achieved.
In addition to validating the process, it may also be necessary
to perform continuous, rigorous microbiological monitoring
during routine production to demonstrate that the
microbiological parameters are within the established
tolerances so as to give an SAL of 10- 6 or better.
In connection with sterilisation by steam, the Z-value relates
the heat resistan ce of a micro-organism to changes in
temperature. The Z-value is the change in temperature
required to alter the D-value by a factor of 10.
The D-value (or decimal reduction value) is the value of a
parameter of sterilisation (duration or absorbed dose)
required to reduce the number of viable organisms to
10 per cent of the original number. It is only of significance
under precisely defined experimental conditions.
The following mathematical relationships apply:
Fo = Dl21 (logNo - logN) = D 121 1og1 F

D I21 D-value ofthe reference spores (5.1.2) at 121 °C,
No initial number of viable micro-organisms,
N final number of viable micro-organisms,
IF inactivation factor.
z= T 2 - TI
logD 1 - logD 2
DI D-value of the micro-organism at temperature TI,

D2 D-value of the micro-organism at temperature T 2 .
IF = No = lOtlD
N
t exposure time,
D D-value of micro-organism in the exposure conditions.
V-A530 Appendix XIX 2014
Coloured glass is obtained by the addition of small amounts of

Appendix XIX metal oxides, chosen according to the desired spectral
absorbance.
Neutral glass is a borosilicate glass containing significant
Containers amounts of boric oxide, aluminium oxide alkali ancl/or
A. Introduction alkaline earth oxides. Due to its composition neutral glass has
This Appendix provides requirements, guidance and information a high hydrolytic resistance and a high thermal shock
on containers for pha17naceutical use. Additional guidance is resistan ce.
provided in a number of B11tish Standards. Attention is drawn in Soda-lime-silica glass is a silica glass containing alkali metal
particular lO British Standards 1679-5:1973, 1679-6:1994, oxides, mainly sodium oxide and alkaline earth oxides,
1679-7:1968 and 1679-8:1992. The expression 'tamper-evident mainly calcium oxide. Due to its composition soda-lime-silica
container' means a closed container fitted with a device that glass has only a moderate hydrolytic resistance.
reveals irreversibly whether the container has been opened, The hydrolytic stability of glass containers for pharrnaceutical
whereas, the expression 'tamper-proof container' means a closed use is expressed by the resistance to the release of soluble
container in which access lO the coments is prevented under normal mineral substances into water under the prescribed
conditions of use. The two terms are considered lO be synonymous conditions of contact between the inner surface of the
by the European Pham¡acopoeia Commission. container or glass grains and water. The hydrolytic resistance
(Ph. Eur. text 3.2) is evaluated by titrating released alkali. According to their
A container for pharrnaceutical use is an artide that contains hydrolytic resistance, glass containers are classified as follows:
or is intended to contain a product and is, or may be, in - Type 1 glass containers: neutral glass, with a high
direct contact with it. The closure is a pan of the container. hydrolytic resistance due to the che mi cal composition of
The container esee General Notices section 1.3) is so the glass itself;
designed that the contents may be removed in a manner - Type II glass containers: usually of soda-lime-silica glass
appropriate to the intended use of the preparation. with a high hydrolytic resistance resulting from suitable
It provides a varying degree of protection depending on the treatment of the surface;
nature of the product and the hazards of the environment, - Type III glass containers: usually of soda-lime-silica glass
and minimises the loss of constituents. The container do es with only moderate hydrolytic resistance.
not interact physically or chemically with the contents in a The following italicised statements constitute general
way that alters their quality beyond the limits tolerated by recommendations conceming the type of glass container that
official requirements. may be used for different types of pharrnaceutical
Single-dose container A single-dose container holds a preparations. The manufacturer of a pharrnaceutical product
quantity of the preparation intended for total or partial use is responsible for ensuring the suitability of the chosen
on 1 occasion only. container.
Multidose container A multidose container holds a Type 1 glass containers are suitable for most preparations whether
quantity of the preparation suitable for 2 or more doses. or not for parenteral use.
Well-closed container A well-dosed container protects Type 11 glass containers are suitable for most acidic and neutral,
the contents from contamination with extraneous solids and aqueous preparations whether or not for paremeral use.
liquids and from loss of contents under ordinary conditions Type 111 glass containers are in general suitable for non-aqueollS
of handling, storage and transporto preparations for parenteral use, for powders for parenteral use
Airtight container An airtight container is impermeable to (except for freeze-dried preparations) and for preparations 110C for
solids, liquids and gases under ordinary conditions of parel1teral use.
handling, storage and transporto If the container is intended Glass containers with a hydrolytic resistance higher than that
to be opened on more than 1 occasion, it must be so recommended aboye for a particular type of preparation may
designed that it remains airtight after re-closure. gene rally also be used.
Sealed container A sealed container is a container closed The container chosen for a given preparation shall be such
by fusion of the material of the container. that the glass material do es not release substances in
Tamper-proof container A tamper-proof container is a quantities sufficient to affect the stability of the preparation
closed container fitted with a device that reveals irreversibly or to present a risk of toxicity. In justified cases, it may be
whether the container has been opened. necessary to have detailed information on the glass
Child-proof container A container that is fitted with a composition, so that the potential hazards can be assessed.
closure that prevents opening by children. Preparations for parenteral administration are normally
presented in colourless glass, but coloured glass may be used
for substances known to be light-sensitive. Colourless or
coloured glass is used for the other pharrnaceutical
B. Glass Containers for Pharmaceutical preparations. It is recommended that all glass containers for
liquid preparations and for powders for parenteral
Use administration permit the visual inspection of the contents.
(Ph. Eur. method 3.2.1 The inner surface of glass containers may be specially treated
Glass containers for pharrnaceutical use are glass articles to improve hydrolytic resistance, to confer water-repellancy,
intended to come into direct contact with pharrnaceutical etc. The outer surface may also be treated, for example ro
preparations. reduce friction and to improve resistance to abrasion.
Colourless glass is highly transparent in the visible spectrum. The outer treatment is such that it does not contaminate the
inner surface of the container.
2014 Appendix XIX B V-A531
Except for type 1 glass containers, glass containers for edge, avoiding overflow and introduction of air bubbles.
pharmaceutical preparations are not to be re-used. Adjust the liquid levels to the brimfulline. Weigh the filled
Containers for human blood and blood components must containers to obtain the mass of the water expressed to 2
not be re-used. decimal places for containers having a nominal volume less
Glass containers for pharmaceutical use comply with the or equal to 30 mL, and expressed to 1 decimal place for
relevant test or tests for hydrolytic resistance. When glass containers having a nominal volume greater than 30 mL.
containers have non-glass components, the tests apply only to Calculate the mean value of the brimful capacity in millilitres
the glass part of the container. and multiply it by 0.9. This volume, expressed to 1 decimal
To define the quality of glass containers according to the place, is the filling volume for the particular container lot.
intended use, one or more of the following tests are Ampoules Place at least 6 dry ampoules on a flat,
necessary. horizontal surface and fill them with distilled water R from a
Tests for hydrolytic resistance are carried out to define the burette, until the water reaches point A, where the body of
type of glass (1, II or III) and to control its hydrolytic the ampoule declines to the shoulder (see Figure 3.2.1.-1).
resistance. Read the capacities (expressed to 2 decimal place s) and
calculate the mean value. This volume, expressed to 1
In addition, containers for aqueous parenteral preparations decimal place, is the filling volume for the particular
are tested for arsenic release and coloured glass containers ampoule lot. The filling volume may also be determined by
are tested for spectral transmission. weighing.
Hyd.rolytic resistance
Table 3.2.1.-1. - Types of glass

Type of container Test to be performed
Type 1 and type II glass containers Test A (surface test)
(to distinguish from type III glass
containers)
Type 1 glass containers (to distinguish Test B (glass grains test) or test e
from type II and type III glass (etching test)
containers) A
Type 1 and type II glass containers Tests A and B, or tests A and e
where it is necessary to determine
whether the high hydrolytic resistan ce
is due to the chemical composition or
to the surface treatment
The test is carried out by titration of the extract solutions

obtained under the conditions described for tests A, B and
C. ,
j
Equipment -~-
- an autoclave capab1e of maintaining a temperature of

121 °C ± 1 °C, equipped with a thermometer or a
Figure 3.2.1.-1. - Filling volume of ampoules (up to point A)
calibrated thermocouple recorder, a pressure gauge, a vent
cock and a tray, of sufficient capacity to accommodate
aboye the water level the number of containers needed to Test A. Hydrolytic resistance of the inner surfaces of
carry out the test; clean che autoclave vessel and all ancillary glass containers (surface test)
equipment choroughly befare use with water R;
The determination is carried out on unused containers.
- burettes with a suitable capacity; The volumes of the test liquid necessary for the final
- one-mark volumetric flasks, with a capacity of 1000 mL; determination are indicated in Table 3.2.1.-2.
- pipettes and beakers;
- conical flasks with a capacity of 100 mL and 250 mL; Table 3.2.1.-2. - Volume of test liquid and number of títrations
- a water-bath;
Filling volume (mL) Volume of test Iiquid Number of titrations
- a metal foil (e.g. aluminium, stainless steel). for one titration (mL)
Flasks and beakers shall' have been already used for the test Up to 3 25.0
or have been filled with water R and kept in an autoclave at
Above 3 and up to 30 50.0 2
121 °C at least for 1 h before being used.
Above 30 and up to 100 100.0 2
Determination of the filling volume
The filling volume is the volume of water to be filled in the Above 100 100.0 3
container for the purpose of the test. For vials and bottles the
filling volume is 90 per cent of the brimful capacity.
For ampoules it is the volume up to the height of the Cleaning Remove any debris or dust. Shortly before the
shoulder. test, rinse each container carefully at least twice with water R
and allow to stand. Immediately before testing empty the
Vials and bottles Select, at random, 6 containers from the
containers, rinse once with water R then with water RI and
sample lot, or 3 if their capacity exceeds 100 mL, and allow to drain. Complete the c1eaning procedure from the
remove any dirt or debris. Weigh the empty containers with first rinsing in not less than 20 min and not more than
an accuracy of 0.1 g. Place the containers on a horizontal
25 mino
surface and fill them with distilled water R until about the rim
V-A532 Appendix XIX B 2014
Heat c10sed ampoules on a water-bath or in an air-oven at Table 3.2.1.-3. - Limil values in the test (or surface hydrolytic
about 50 oC for approximately 2 min before opening; do not resistance
rinse before testing. Maximum volume of 0.01 M Hel per
100 mL of test liquid (mL)
Filling and heating The containers are filled with Glass containers
water Rl up to the filling volume. Containers in the form of
Filling volume (mL) Types 1 and 11 Type III
cartridges or prefilled syringes are c10sed in a suitable
manner with material that do es not interfere with the test. Up lo 1 2.0 20.0
Each container including ampoules shall be loosely capped Above 1 and up lo 2 1.8 17.6
with an inert material such as a dish of neutral glass or
Aboye 2 and up lo 5 1.3 13.2
aluminium foil previously rinsed with water R. Place the
containers on the tray of the autoclave. Place the tray in the Aboye 5 and up lo 10 1.0 10.2
autoclave containing a quantity of water R such that the tray Aboye 10 and up lo 20 0.80 8.1
remains c1ear of the water. Close the autoclave and carry out
Above 20 and up lo 50 0.60 6. 1
the following operations:
- heat the autoclave to 100 oC and allow the steam to issue Aboye 50 and up lo 100 0.50 4.8
from the vent cock for 10 min; Aboye 100 and up lo 200 0.40 3.8
- c10se the ventcock and raise the temperature from 100 oC Aboye 200 and up lO 500 0.30 2.9
to 121 °C at arate of 1 oC per min;
Aboye 500 0.20 2.2
- maintain the temperature at 121 ± 1 oC for 60 ± 1 min;
- Iower the temperature from 121 °C to 100 oC at arate of
0.5 oC per min, venting to prevent vacuum; - a set of 3 square-mesh sieves of stainless steel, mounted
- do not open the autoclave before it has cooled down to on frames of the same material and consisting of the
95 oC; following:
- remove the containers from the autoclave using normal Ca) sieve no. 710;
precautions, place them in a water-bath at 80 oC, and run Cb) sieve no. 425;
cold tap water, taking care that the water does not contact Cc) sieve no. 300;
the loose foil caps to avoid contamination of the - a permanent magnet;
extraction solution;
- a metal foil Ce.g. aluminium, stainless steel);
- cooliI).g time does not exceed 30 min.
- a hot-air oven, capable of maintaining a temperature of
The extraction solutions are analysed by titration according 140 ± 5 oC;
to the method described below.
- a balance, capable of weighing up to 500 g with an
Method Carry out the titration within 1 h of removal of accuracy of 0.005 g;
the containers from the autoclave. Combine the liquids
obtained from the containers and mix. Introduce the - a desiccator;
prescribed volume CTable 3.2.l.-2) into a conical fiask. Place - an ultrasonic bath.
the same volume of water Rl into a second similar fiask as a Method Rinse the containers to be tested with water R and
blank. Add to each fiask 0.05 mL of methyl red solution R for dry in the oven. Wrap at least 3 of the glass articles in c1ean
each 25 mL of liquid. Titrate the blank with 0.01 M paper and crush to produce 2 samples of about 100 g each
hydrochloric acid. Titrate the test liquid with the same acid in pieces not more than 30 mm across. Place 30-40 g of the
until the colour of the resulting solution is the same as that pie ces between 10-30 mm across taken from 1 of the
obtained for the blank. Subtract the value found for the samples in the mortar, insert the pestle and strike it heavily
blank titration from that found for the test liquid and express once only with the hammer. Transfer the contents of the
the results in millilitres of 0.01 M hydrochloric acid per mortar, to the coarsest sieve Ca) of the set. Repeat the
100 mL. Express titration values of less than 1.0 mL to 2 operation until all fragments have been transferred to the
decimal place s and titration values of more than or equal to sieve. Shake the set of sieves a short time by hand and
1.0 mL to 1 decimal place. remove the glass which remains on sieves Ca) and Cb).
Limits The results, or the average of the results if more Submit these portions to further fracture, repeating the
than one titration is performed, is not greater than the values operation until about 10 g of glass remains on sieve Ca).
stated in Table 3.2 .l.-3 . Reject this portion and the portion which passes through
sieve Cc). Reassemble the set of sieves and shake for 5 mino
Test B. Hydrolytic resistance 01 glass grains (glass Transfer to a weighing bottle those glass grains which passed
grains test)
through sieve Cb) and are retained on sieve Cc). Repeat the
Check that the articles as received have been annealed lO a crushing and sieving procedure with the other glass simple
commercially acceptable quality. and thus 2 samples of grains, each of which shall be in
The test may be performed on the canes used for the excess of 10 g, are obtained. Spread each sample on a piece
manufacture of tubing glass containers or on the containers. of c1ean glazed paper and remove any iron particles by
EQUIPMENT passing the magnet over them. Transfer each sample into a
- a mortar, pestle Csee Figure 3.2.l.-2) and hammer in beaker for c1eaning. Add to the grains in each beaker 30 mL
tempered, magnetic steel; of acewne R and scour the grains by suitable means, such as
a rubber or plastic-coated glass rod . After scouring the
grains, allow to settle and decant as much acetone as
possible. Add another 30 mL of acewne R, swirl, decant
again and add a new portion of acewne R .
2014 Appendix XIX B V-A533
NOTE: where necessary to obtain a sharp end-point, the clear

solution is to be decanted into a separate 250 mL fiask. Rinse the
grains with 3 quantities, each 01 15 mL, 01 water Rl by swirling
and add the washings to the main solution. Add 0.05 mL 01 the
methyl red solution R. Titrate and calculate as descn'bed below.
In this case also add 45 mL 01 water Rl and 0. 05 mL 01 methyl
red solution R to the blank solution.
Calculate the mean value of the results in millilitres of
0.02 M hydrochloric acid per gram of the sample and if
required its equivalent in alkali extracted, calculated as
micrograms of sodium oxide per gram of glass grains.
1 mL of 0.02 M hydrochloric acid is equivalent to 620 flg of
sodium oxide.
Repeat the test if the highest and lowest observed values
differ by more than 20 per cent.
Limits Type I glass containers require not more than
0.1 mL of 0.02 M hydrochloric acid per gram of glass, type n
and type nI glass containers require not more than 0.85 mL
of 0.02 M hydrochloric acid per gram of glass.
Test C. To determine whether the containers have
been surface treated (etching test)
When it is necessary to determine if a container has been
surface-treated, andlor distinguish between type I and type n
glass containers, test C is used in addition to test A.
Altematively, test A and B may be used. Test C may be
carried out either on unused samples or on samples
Figure 3.2.1.-2. - Apparatus for glass grains method previously tested for test A.
Ydimensions in millimetres) Vials and botdes The volumes of test liquid required are
shown in Table 3.2.1.-2.
Fill the bath of the ultrasonic vessel with water at room Rinse the containers twice with water R and fill to the brimful
temperature, then place the beaker in the rack and irnmerse point with a mixture of 1 volume of hydrofiuoric acid R and
it until the level of the acetone is at the level of the water; 9 volumes of hydrochloric acid R and allow to stand for
apply the ultrasound for 1 mino Swirl the beaker, allow to 10 mino Empty the containers and rinse carefully 5 times
settle and decant the acetone as completely as possible and with water R. Immediate1y before the test, rinse once again
then repeat the ultrasonic cleaning operation. If a fine with water R. Submit the containers thus prepared to the
turbidity persists, repeat the ultrasonic cleaning and acetone same autoclaving and determination procedure as described
washing until the solution remains clear. Swirl and decant in test A for surface hydrolytic resistance. If the results are
the acetone then dry the grains, first by putting the beaker on considerably higher than those obtained from the original
a warm plate to remove excess acetone and then by heating surfaces (by about a factor of 5 to 10), the samples have
at 140 oC for 20 min in the drying oven. Transfer the dried been surface-treated.
grains from each beaker into separate weighing bottles, insert Ampoules
the stoppers and cool in the desiccator. Weigh 10.00 g of the NOTE: ampoules made from glass tubing are not normally
cleaned and dried grains into 2 separate conical fiasks. subjected to internal sulface treatment because their high chemical
Add 50 mL of water Rl into each by means of a pipette (test resistance is dependent upon the chemical composition of the glass
solutions). Pipette 50 mL of water Rl into a third conical as a material.
fiask which will serve as a blank. Distribute the grains evenly
Apply the test method as described aboye for vials and
over the fiat bases of the fiasks by gentle shaking. Close the
bottles. If the ampoules are not surface-treated, the new
fiasks with neutral glass dishes or aluminium foil rinsed with
values' are slightly lower than those obtained in previous tests.
water R or with inverted beakers so that the inner surface of
the beakers fit snugly down onto the top rims of the fiasks. Distinction between Type 1 and Type 11 glass
Place all 3 fiasks in the rack in the autoclave containing the containers
water at ambient temperature, and ensure that they are held The results obtained in Test C are compared to those
aboye the level of the water in the vessel. Carry out the obtained in Test A. The interpretation of the result is shown
autoclaving procedure in a similar manner to that described in Table 3.2.1.-4.
under test A, but maintain the temperature of 121 ± 1 oC
only for 30 ± 1 mino Do not open the autoclave until it has Table 3.2.1.-4. - Distinclion between Types 1 and 11 glass
cooled to 95 oC. Remove the hot samples from the autoclave containers
and cool the fiasks in running tap water as soon as possible, Type 1 Type II
avoiding thermal shock. To each of the 3 fiasks add 0.05 mL
of methyl red solution R . Titrate the blank solution The values are c10sely similar to The values greatly exceed those lound in
those found in the test lor surface the test for surface hydrolytic resistance
immediately with 0.02 M hydrochloric acid then titrate the test hydrolytic resistance for type 1 and are similar but not larger than
solutions until the colour matches that obtained with the glass containers. those for type III glass containers.
blank solution. Subtract the titration volume for the blank
solution from that for the test solutions.
V-A534 Appendix XIX B 2014
Arsenic containers for parenteral preparations do es not exceed the

The test applies to glass containers for aqueOltS parenteral limits given in Table 3.2.1.-5.
preparations.
Hydride generation aromic absorption spectrometry (2.2.23, Table 3.2.1.-5. - Limits of specfral transmission for coloured
Method I). glass containers for parenteral preparations
Test solution Use the extract solution obtained from Maximum percentage of spectral Iransmis.ion al
any wavelenglh between 290 nm and 450 nm
containers of types I and II, after autoc1aving at 121 oC for
1 h as described under test A for surface hydrolytic Nominal volume (mL) Flame·sealed conlainers Containers wilh
closures
resistance. Transfer 10.0 mL ro a 100 mL volumetric ftask.
Add 10 mL of hydrochloric acid R and 5 mL of a 200 gIL Up lo 1 50 25
solution of potassium iodide R. Heat on a water-bath at 80 oC Above 1 and up lo 2 45 20
for 20 min, allow to cool and dilute to 100.0 mL with
Above 2 and up lo 5 40 15
water R.
Above 5 and up lo 10 35 13
Reference solutions Prepare the reference solutions using
arsenic standard solurion (1 ppm As) R. Add 10 mL of Above 10 and up lo 20 30 12
hydrochloric acid R and 5 mL of a 200 gIL solution of Above 20 25 10
potassium iodide R. Heat on a water-bath at 80 oC for
20 min, allow ro cool and dilute to 100.0 mL with water R.
The concentration range of the reference solutions is
typically 0.005 ppm ro 0.015 ppm of As. Annex - test for surface hydrolytic resistance -
determination by ftame atornic absorption
Acid reservoir Hydrochloric acid R.
spectrometry (FAAS)
Reducing reservoir Sodium tetrahydroborate reducing
The suiface hydrolytic resistance of glass of types 1 and JI may be
solution R.
determined by analysis of the leaching solutian by fiame atomic
Use a hydride generation device to introduce the test solution absorprion spectrometry. A number of elements that, when present
into the cuvette of an aromic absorption spectrometer. as oxides in glass, contribute to the alkalinity of the solution, are
Establish and standardise instrumental operating conditions detennined and used to express an alkali equivalent.
according to the manufacturer's instructions, optimise the The spectrometric method has the advantage of allowing the use of
uptake rate 'of the peristaltic pump tubings, then connect a much smaller sample of extraer so that it can be applied to small
tubings to the acid reservo ir, the reducing reservoir and the individual containers. This enables an evaluation of the unijonnity
test solution. of the containers in a given batch where this is critical. The resules
Source Hollow-cathode lampo of this measurement are not equivalent to those of titrimetry and
Wavelength 193.7 nm, the 2 methods cannot be considered interchangeable. A correlation
Atomisation device Air-acetylene flameo between the 2 is dependent on the type of glass and the size and
shape of the container. The tim'metric method is the reference
Limit Maximum 0.1 ppm of As.
method of the Pharmacopoeiaj the spectrometric method may be
Spectral transmission for coloured glass containers used in justified and authorised cases.
Equipment A UV-VIS spectrophotometer, equipped with A method suitable for this type of analysis is shown below.
a photodiode detector or equipped with a photomultiplier The determination is carried out on unused containers.
tube coupled with an integrating sphere. The number of containers to be examined is indicated in
Preparation of the specimen Break the glass container Table 3.2.1.-6,
or cut it with a circular saw fitted with a wet abrasive wheel,
such as a carborundum or a bonded-diamond wheel. Select Table 3.2.1.-6. - Number of containers to be examined for fhe
sections representative of the wall thickness and trim them as spectromefric method
suitable for mounting in a spectrophotometer. If the Filling volume (mL) Number of containers lo Addilional container.
specimen is too small to cover the opening in the specimen be measured separalely for preliminary
measurements
holder, mas k the uncovered portion with opaque paper or
tape, provided that the length of the specimen is greater than Up to 2 20 2
that of the slit. Before placing in the holder, wash, dry and Above 2 and up lo 5 15 2
wipe the specimen with lens tissue. Mount the specimen
Abov. 5 and up lo 30 10
with the aid of wax, or by other convenient mean s, taking
care to avoid leaving fingerprints or other marks. Above 30 and up lo 100 5
Method Place the specimen in the spectrophotometer with Above 100 3
its cylindrical axis parallel to the slit and in such a way that
the light beam is perpendicular to the surface of the section
and that the losses due to reftection are at a minimum. Instructions on determination of the filling volume, c1eaning
Measure the transmission of the specimen with reference to of the containers, filling and heating are given aboye under
air in the spectral region of 290-450 run, continuously or at Hydrolytic resistance and Test A. Hydrolytic resistance of the
intervals of 20 nm. inner surfaces of glass containers.
Limits The observed spectral transmission for coloured Solutions
glass containers for preparations that are not for parenteral Spectrochemical buffer solution Dissolve 80 g of
administration does not exceed 10 per cent at any caesium ch/oride R in about 300 mL of water RI, add 10 mL
wavelength in the range of 290 nm to 450 nm, irrespective of 6 M hydrochloric acid R and transfer to a 1000 mL
of the type and the capacity of the glass container. volumetric flask. Dilute to volume with water RI and mix.
The observed spectral transmission in coloured glass
2014 Appendix XIX e V-A535
Stock solutions: sodium oxide per millilitre of the extraction solution using
- sodium oxide, c(NazO) = 1 mg/mL; the following mass conversion factors:
- potassium oxide, c(K2 0) = 1 mg/mL; - 1 flg of potassium oxide corresponds to 0.658 ~lg of
sodium oxide;
- calcium oxide, c(CaO) = 1 mg/mL.
- 1 flg of calcium oxide corresponds to 1.105 flg of sodium
Commercially available stock solutions may also be used .
oxide.
Standard solutions Prepare standard solutions by diluting
Limits For each container tested, the result is not greater
the stock solutions with water Rl to obtain concentrations
suitable for establishing the reference solutions in appropriate than the value given in Table 3.2.l.-7.
manner, e.g. with concentrations of 20 flg/mL of sodium
oxide, potassium oxide and calcium oxide, respectively. Table 3.2.1.-7. - Limit values in the test for surface hydrolytic
Commercially available standard solutions may also be used. resistan ce by flame atomic absorption spectrometry
Reference solutions Prepare the reference solutions for Maximum values for the
concentration of oxides, expressed
establishing the calibration graph (set of calibration as sodium oxide (iJg/mL)
solutions) by diluting suitable concentrated standard Filling volume (mL) Glass containers
solutions with water Rl, so that the normal working ranges
Types 1 and II
of the specific elements are covered, taking into account the
instrument used for the measurement. Typical concentration Up to 1 5.00
ranges of the reference solutions are: Aboye 1 and up to 2 4.50
- for determination by atomic emission spectrometry of Above 2 and up to 5 3.20
sodium oxide and potassium oxide: up to 10 flg/mL;
Aboye 5 and up to 10 2.50
- for determination by atomic absorption spectrometry of
sodium oxide and potassium oxide: up to 3 flg/mL; Aboye 10 and up to 20 2.00
- for determination by atomic absorption spectrometry of Aboye 20 and up to 50 1.50

calcium oxide: up to 7 flg/mL. Aboye 50 and up to 100 1.20
- Use reference solutions containing 5 per cent V/V of the Above 100 and up to 200 1.00
spectrochemical buffer solution.
Aboye 200 and up to 500 0.75
Method Aboye 500 0.50
Carry out preliminary measurements of the potassium oxide
and calcium oxide concentrations on one of the extraction
solutions. If, fot one container type, the concentration of
potassium oxide is less than 0.2 flg/mL and if the
concentration of calcium oxide is less than 0.1 flg/mL, the
remaining extraction solutions of this container type need not C. Plastic Containers and Closures
be analysed for these ions. Aspirate the extraction solution
from each sample directly into the flame of the atomic
A plastic container for pharmaceutical use is a plastic artiele
absorption or atomic emission instrument and determine the
which contains or is intended to contain a pharmaceutical
approximate concentrations of sodium oxide (and potassium
product and is, or may be, in direct contact with it.
oxide and calcium oxide, if present) by reference to
The elosure is a part of the container.
calibration graphs produced from the reference solutions of
suitable concentration. Plastic containers and elosures for pharmaceutical use are
made of materials in which may be ineluded certain
Final determination additives; these materials do not inelude in their composition
If dilution is unnecessary add to each container a volume of any substance that can be extracted by the contents in such
the spectrochemical buffer solution equivalent to 5 per cent quantities as to alter the efficacy or the stability of the
of the filling volume, mix well and determine sodium oxide, product or to present a risk of toxicity.
calcium oxide and potassium oxide, if present, by reference The most commonly used polymers are polyethylene (with
to calibration graphs. For the determination of the calcium and without additives), polypropylene, poly(vinyl chloride),
oxide concentration by flame atomic spectrometry, the poly(ethylene terephthalate) and poly(ethylene-vinyl acetate).
nitrous oxide/acetylene flame shall be used.
The nature and amount of the additives are determined by
If dilution is necessary, determine sodium oxide, calcium the type of the polymer, the process used to convert the
oxide and potassium oxide, if present, following the polymer into the container and the intended purpose of the
pro ce dures as described aboye. The measuring solutions shall container. Additives may consist of antioxidants, stabilisers,
contain 5 per cent V/V of the spectrochemical buffer plasticisers, lubricants, colouring matter and impact
solution. Concentration values les s than 1.0 flg/mL are modifiers. Antistatic agents and mould-release agents may be
expréssed to 2 decimal places, values greater than or equal to used only for containers for preparations for oral use or for
1.0 flg/mL to 1 decimal place. Correct the result for the external use for which they are authorised. Acceptable
buffer addition and for dilution, if any. additives are indicated in the type specification for each
Calculation material described in the Pharmacopoeia. Other additives
Calculate the mean value of the concentration of individual may be used provided they are approved in each case by the
oxides found in each of the samples tested, in micrograms of competent authority responsible for the licensing for sale of
the oxide per millilitre of the extraction solution and calculate the preparation.
the sum of the individual oxides, expressed as micrograms of For selection of a suitable plastic container, it is necessary to
know the full manufacturing formula of the plastic, ineluding
al! materials added during formation of the container so that
V-A536 Appendix XIX e 2014
the potential hazards can be assessed. The plastic container a part that allows them to be suspended and which will
chosen for any particular preparation should be such that: withstand the tension occurring during use. The containers
- the ingredients of the preparation in contact with the must withstand the sterilisation conditions to which they will
plastic material are not significantly adsorbed on its be submined. The design of the container and the method of
surface and do not significantly migra te into or through sterilisation chosen are such that all parts of the containers
the plastic, that may be in contact with the infusion are sterilised.
The containers are impermeable to micro-organisms after
- the plastic material does not release substances in
closure. The containers are such that after filling they are
quantities sufficient to affect the stability of the
resistant to damage from accidental freezing which may occur
preparation or to present a risk of toxicity.
during transport of the final preparation. The containers are
Using material or materials selected to satisfy these criteria, a and remain sufficiently transparent to allow the appearance
number of identical type samples of the container are made of the contents to be examined at any time, unless otherwise
by a well-defined procedure and submined to practical justified and authorised.
testing in conditions that reproduce those of the intended
The empty containers display no defects that may lead to
use, including, where appropriate, sterilisation. In order to
leakage and the filled and closed containers show no leakage.
confirm the compatibility of the container and the contents
and to ensure that there are no changes detrimental to the For satisfactory storage of sorne preparations, the container
quality of the preparation, various tests are carried out such has to be enclosed in a protective envelope. The initial
as verification of the absence of changes in physical evaluation of storage has then to be carried out using the
characteristics, assessment of any loss or gain through container enclosed in the envelope.
permeation, detection of pH changes, assessment of changes
caused by light, chemical tests and, where appropriate, Tests
biological tests. Solution S Use solution S within 4 h of preparation. Fill a
The method of manufacture is such as to ensure container to its nominal capacity with water R and close it, if
reproducibility for subsequent bulk manufacture and the possible using the usual means of closure; otherwise close
conditions of manufacture are chosen so as to preclude the using a sheet of pure aluminium. Heat in an autoclave so
possibility of contamination with other plastic materials or that a temperature of 121 ± 2 oC is reached within 20 min
their ingredients. The manufacturer of the product must to 30 min and maintain at this temperature for 30 mino
ensure that containers made in production are similar in If heating at 121 °C leads to deterioration of the container,
every respect to the type samples. heat at 100 oC for 2 h.
For the results of the testing on type samples to remain valid, Blank Prepare a blank by heating water R in a borosilicate-
it is important that: glass flask closed by a sheet of pure aluminium at the
temperature and for the time used for the preparation of
- there is no change in the composition of the material as
solution S.
defined for the type samples,
Appearance of solution S Solution S is clear (2.2.1) and
- there is no change in the manufacturing process as
colourless (2.2.2, Method IJ) .
defined for the type samples, especially as regards the
temperatures to which the plastic material is exposed Acidity or aIkalinity To a volume of solution S
during conversion or subsequent procedures such as corresponding to 4 per cent of the nominal capacity of the
sterilisa tion, container add 0.1 mL of phenolphthalein solution R.
The solution is colourless. Add 0.4 mL of 0.01 M sodium
- scrap material is not used.
hydroxide. The solution is pink. Add 0.8 mL of 0.01 M
Recycling of excess material of well-defined nature and hydroehlorie acid and 0.1 mL of methyl red solution R .
proportions may be permined after appropriate validation. The solution is orange-red or red.
Subject to satisfactory testing for compatibility of each Absorbance (2.2.25) Measure the absorbance of solution
different combination of container and contents, the S from 230 nm to 360 nm, using the blank (see solution S)
materials described in the Pharmacopoeia are recognised as as the compensation liquido At these wavelengths, the
being suitable for the specific purposes indicated, as defined absorbance is not greater than 0.20.
aboye.
Reducing substances To 20.0 mL of solution S add
Plastic containers for aqueous solutions for 1 mL of dilute sulfurie acid R and 20.0 mL of 0.002 M
parenteral infusion potassium permanganate. Boil for 3 mino Cool immediately.
Add 1 g of potassium iodide R and titrate immediately with
(Ph. Eur text 3.2.2.1 NOTE: This supersedes the text 3.2.7) 0.01 M sodium thiosulfate, using 0.25 mL of stareh solution R
as indicator. Carry out a titration using 20.0 mL of the
Definition
blank. The difference between the titration volumes is not
Plastic containers for aqueous solutions for infusion are greater than 1.5 mL.
manufactured from one or more polymers, if necessary with
Transparency Fill a container previously used for the
additives. The containers described in this section are not
preparation of solution S with a volume equal to the nominal
nec'essarily suitable for emulsions. The polymers most
capacity of the primary opalescent suspension (2.2.1) diluted
commonly used are polyethylene, polypropylene and
1 in 200 for a container made from polyethylene or
poly(vinyl chloride) . The specifications 'of this text are to be
polypropylene and 1 in 400 for other containers.
read in conjunction with section 3.2.2. Plastíe eontainers and
The cloudiness of the suspension is perceptible when viewed
closures for pharmaeeutical use.
through the container and compared with a similar container
The containers may be bags or bonles. They have a site filled with water R.
suitable for the anachment of an infusion set designed to
ensure a secure connection. They may have a site that allows
an injection to be made at the time of use. They usually have
2014 Appendix XIX D V-A537
Labelling Characters
The label accompanying a batch of empty containers The container is sufficiently transparent to allow adequate
includes a statement of: visual examination of its contents before and after the taking
- the name and address of the manufacturer, of the blood and is sufficiently flexible to olfer minimum
resistance during fining and emptying under normal
- a batch number wruch enables the history of the container
conditions of use. The container contains not more than
and of the plastic material of which it is manufactured to
5 mL of air.
be traced.
Tests
Solution SI Fill the container with 100 mL of a sterile,
D. Containers tor Blood and Blood pyrogen-free 9 gIL solution of sodium chloride R. Close the
container and heat it in an autoclave so that the contents are
Components maintained at 110 oC for 30 mino
If the container to be examined contains an anticoagulant
1. Sterile Plastic Containers for Human Blood and
solution, first empty it, rinse the container with 250 mL of
Blood Components
water for injections R at 20 ± 1 oC and discard the rinsings.
(Ph. Eur. method 3.2.3) Solution S2 Introduce into the container a volume of water
Plastic containers for the collection, storage, processing and for injections R corresponding to the intended volume of
administration of blood and its components are anticoagulant solution. Close the container and heat it in an
manufactured from one or more polymers, if necessary with autoclave so that the contents are maintained at 110 oC for
additives . The composition and the conditions of 30 mino After cooling, add sufficient water for injections R to
manufacture of the containers are registered by the fin the container to its nominal capacity.
appropriate competent authorities in accordance with the
If the container to be examined contains an anticoagulant
relevant national legislation and international agreements.
solution, first empty it and rinse it as indicated aboye.
When the composition of the materials of the different parts
Resistance to centrifugation Introduce into the container
of the containers correspond to the appropriate specifications,
a volume of water R, acidified by the addition of 1 mL of
their quality is controlled by the methods indicated in those
di/ute hydrochloric acid R, sufficient to fill it to its nominal
specifications (see 3.1. Materials usedfor the manufacture of
capacity. Envelop the container with absorbent paper
containers and subsections).
impregnated with a 1 in 5 dilution of bromophenol blue
Materials other than those described in the Pharmacopoeia solution Rl or other suitable indicator and then dried.
may be used provided that their composition is authorised by Centrifuge at 5000 g for 10 mino No leakage perceptible on
the competent authority and that the containers the indicator paper and no permanent distortion occur.
manufactured from them comply with the requirements
Resistance to stretch Introduce into the container a
prescribed for Sterile Plastic Containers for Human Blood
volume of water R, acidified by the addition of 1 mL of di/ute
and Blood Components.
hydrochloric acid R, sufficient to fin it to its nominal capacity.
In normal conditions of use the materials do not release Suspend the container by the suspending device at the
monomers, or other substances, in amounts likely to be opposite end from the blood-taking tube and apply along the
harrnful nor do they lead to any abnormal modifications of axis of this tube an immediate force of 20 N (2.05 kg±) .
the blood. Maintain the traction for 5 s. Rep eat the test with the force
The containers may contain anticoagulant solutions, applied to each of the parts for fining and emptying.
depending on their intended use, and are supplied sterile. No break and no deterioration occur.
Each container is fitted with attachments suitable for the Leakage Place the container which has been submitted to
intended use. The container may be in the form of a single the stretch test between two plates coveréd with absorbent
unit or the collecting container may be connected by one or paper impregnated with a 1 in 5 dilution of bromophenol blue
more tubes to one or more secondary containers to allow solution Rl or other suitable indicator and then dried.
separation of the blood components to be effected within a Progressively apply force to the plates to press the container
closed system. so that its internal pressure (i.e. the difference between the
The outlets are of a shape and size allowing for adequate applied pressure and atrnospheric pressure) reaches 67 kPa
connection of the container with the blood-giving equipment. within 1 mino Maintain the pressure for 10 mino No signs of
The protective coverings on the blood-taking needle and on leakage are detectable on the indicator paper or at any point
the appendages must be such as to ensure the maintenance of attachment (seals, joints, etc.).
of sterility. They must be easily removable but must be Vapour permeability For a container containing an
tamper-proof. anticoagulant solution, fin with a volume of a 9 gIL solution
The capacity of the containers is related to the nominal of sodium chloride R equal to the volume of blood for which
capacity prescribed by the national authorities and to the the container is intended.
appropriate volume of anticoagulant solution. The nominal For an empty container, fill with the same mixture of
capacity is the volume of blood to be collected in the anticoagulant solution and sodium chloride solution. Close
contailter. The containers are of a shape such that when the container, weigh it and store it at 5 ± 1 oC in an
fined they may be centrifuged. atrnosphere with a relative humidity of (50 ± 5) per cent for
The containers are fitted with a suitable device for 21 days. At the end of this period the loss in mass is not
suspending or fixing which does not hinder the collection, greater than 1 per cent.
storage, processing or administration of the blood. Emptying under pressure Fill the container with a
The containers are enclosed in sealed, protective envelopes . volume of water R at 5 ± 1 oC equal to the nominal
capacity. Attach a transfusion set without an intravenous
V-A538 Appendix XIX D 2014
cannula to one of the connectors. Compress the container so 4.75 mL of buffer solution Bo and 10.25 mL of water R
as to maintain throughout the emptying an internal pressure (solution C l ).
(i.e the difference between the applied pressure and To tubes I, U and UI add, respectively, 1.5 mL of solution
atmospheric pressure) of 40 kPa. The container empties in Al, 1.5 mL of solution BI and 1.5 mL of solution C I . At the
less than 2 mino same time and in the same manner, prepare three other
Speed of filling Attach the container by means of the tubes, replacing solution S2 by water R. Centrifuge
blood-taking tube fitted with the needle to a reservoir simultaneously the tubes to be examined and the control
containing a suitable solution having a viscosity equal to that tubes at exactly 2500 g in the same horizontal centrifuge for
of blood, such as a 335 gIL solution of sucrose R at 37 oc. 5 mino After centrifuging, measure the absorbances (2.2.25)
Maintain the internal pressure of the reservoir of the liquids at 540 nm using the stock buffer solution as
(i.e. the difference between the applied pressure and compensation liquido Calculate the haemolytic value as a
atmospheric pressure) at 9.3 kPa with the base of the percentage from the expression:
reservoir and the upper part of the container at the same
leve!. The volume of liquid which fiows into the container in
Aexp x 100
8 min is not less than the nominal capacity of the container. A 100
Resistance to temperature variations Place the
container in a suitable chamber having an initial temperature A lOO absorbance of tube IU,
of 20-23 oc. Cool it rapidly in a deep-freeze to - 80 oC and A exp absorbance of tube I or U or of the corresponding
maintain it at this temperature for 24 h. Raise the control tubes.
temperature to 50 oC and maintain for 12 h . AlIow to cool
to room temperature. The container complies with the tests The solution in tube I gives a haemolytic value not greater
for resistance to centrifugation, resistance to stretch, leakage, than 10 per cent and the haemolytic value of the solution in
vapour permeability emptying under pressure and speed of tube U does not differ by more than 10 per cent from that of
filling prescribed aboye. the corresponding control tube.
Transparency Fill the empty container with a volume Sterility (2.6.1) The containers comply with the test for
equal to its nominal capacity of the primary opalescent sterility. Introduce aseptically into the container 100 mL of a
suspension (2.2.1) diluted so as to have an absorbance sterile 9 gIL solution of sodium chloride and shake the
(2.2.25) at 640 nm of 0.37 to 0.43 (dilution factor about 1 container to ensure that the internal surfaces have been
in 16) . The cloudiness of the suspension must be perceptible entirely wetted. Filter the contents of the container through a
when viewed through the bag, as compared with a similar membrane filter and place the membrane in the appropriate
container filled with water R. culture medium, as prescribed in the test for sterility.
Extractable matter Tests are carried out by methods Pyrogens (2.6.8) Solution SI complies with the test for
designed to simulate as far as possible the conditions of pyrogens. Inject 10 mL of the solution per kilogram of the
contact between the container and its contents which occur rabbit's mass.
in conditions of use. Abnormal toxicity (2.6.9) Solution SI complies with the
The conditions of contact and the tests to be carried out on test for abnormal toxicity. Inject 0.5 mL of the solution into
the eluates are prescribed, according to the nature of the each mouse.
constituent materials, in the particular requirements for each
type .of container. Packaging
HAEMOLYTIC EFFECTS IN BUFFERED SYSTEMS The containers are packed in protective envelopes.
Stock buffer ~olution Dissolve 90.0 g of sodium chloride R, On removal from its protective envelope the container shows
34.6 g of disodium hydrogen phosphate R and 2.43 g of sodium no leakage and no growth of micro-organisms. The protective
dihydrogen phosphate R in water R and dilute to 1000 mL envelope is sufficiently robust to withstand normal handling.
with the same solvento The protective envelope is sealed in such a manner that it
Buffer solution Ao To 30.0 mL of stock buffer solution cannot be opened and re-closed without leaving visible traces
add 10.0 mL of water R. that the seal has been broken.
Buffer solution Bo To 30.0 mL of stock buffer solution
Labelling
add 20.0 mL of water R.
The labelling complies with the relevant nationallegislation
Buffer solution Co To 15.0 mL of stock buffer solution
and international agreements. The label states:
add 85.0 mL of water R.
- the name and address of the manufacturer,
Introduce 1.4 mL of solution S2 into each of three centrifuge
tubes. To tube I add 0.1 mL of buffer solution Ao, to tube U - a batch number which enables the history of the container
add 0.1 mL of buffer solution Bo and to tube III add 0.1 mL and of the plastic material of which it is manufactured to
ofbuffer ~olution C o. To each tube add 0.02 mL offresh, be traced.
heparinised human blood, mix well and warm on a water- A part of the label is reserved for:
bath at 30 ± 1 oC for 40 mino Use blood collected less than - the statement of the blood group, the reference number
3 h previously or blood collected into an anticoagulant and all other information required by nationallegislation
citrate-phosph~te-dextrose solution (CPD) less than 24 h or international agreements, and an empty space is
previously. provided for the insertion of supplementary labelling.
Prepare three solutions containing, respectively: The label of the protective envelope or the labe! on the
3.0 mL of buffer solution Ao and 12.0 mL of water R container, visible through the envelope, states:
(solution Al), - the expiry date,
4.0 mL of buffer solution Bo and 11 .0 mL of water R - that, once withdrawn from its protective envelope, the
(solution B l ), container must be used within 10 days.
2014 Appendix XIX D V-A539
The ink or other substance used to print the labels or the equal to half the nominal volume with the extraction solvent,
writing must not diffuse into the plastic material of the previously heated to 37 oC in a well-stoppered fiask. Expel
container and must remain legible up to the time of use. the air completely from the container and seal the donor
tube. Immerse the filled container in a horizontal position in
2. Empty Sterile Containers of Plasticised a water-bath maintained at 37 ± 1 oC for 60 ± 1 min
Poly(Vinyl Chloride) for Human Blood and Blood without shaking. Remove the container from the water-bath,
Components invert it gently 10 times and transfer the contents to a glass
(Ph. Eur. method 3.2.4) fiask. Immediately measure the absorbance at the maximum
Unless otherwise authorised as described under Sterile plastic at 272 nm, using the extraction solvent as compensation
containers jor human blood and blood components (3.2.3), the liquido
nature and composition of the material from which the Determine the concentration of di(2-ethylhexyl) phthalate in
containers are made comply with the requirements for milligrams per 100 mL of extract from the calibration curve.
Materials based on plasticised poly(vinyl chloride) jor containers The concentration do es not exceed:
jor human blood and blood components and jor containers jor - 10 mg per 100 mL for containers of nominal volume
aqueous solutions jor intravenous injusion (3. 1. 1). greater than 300 mL but not greater than 500 mL;
Tests - 13 mg per 100 mL for containers of nominal volume
greater than 150 mL but not greater than 300 mL;
They comply with the tests prescribed for Sterile plastic
containers jor human blood and blood components (3.2.3) and - 14 mg per 100 mL for containers of nominal volume up
with the following tests to detect extractable matter. to 150 mL.
Reference solution Heat water jor injections R in a Chlorides (2.4.4) Maximum 0.4 ppm, determined with
borosilicate-glass fiask in an autoclave at 110 oC for 30 mino solution S2'
Acidity or alkalinity To a volume of solution Sz Prepare the standard using a mixture of 1.2 mL of chloride
corresponding to 4 per cent of the nominal capacity of the standard solution (5 ppm el) R and 13.8 mL of water R.
container add 0.1 mL of phenolphthalein solution R. Arnmonium (2.4.1) Maximum 2 ppm.
The solution remains colourless. Add 0.4 mL of 0.01 M Dilute 5 mL of solution S2 to 14 mL with water R .
sodium hydroxide. The solution is pink. Add 0.8 mL of Residue on evaporation Evaporate to dryness 100 mL of
0.01 M hydrochloric acid and 0.1 mL of methyl red solution R. solution S2 in a borosilicate-glass beaker of appropriate
The solution is orange-red or red. capacity, previously heated to 105 oc. Evaporate to dryness
Absorbance (2.2.25) Maximum 0.3 0, determined between in the same conditions 100 mL of the reference solution
wavelengths of 230 nm and 250 nm on solution Sz; (blank test). Dry to constant mass at 100-105 oC.
maximum 0.10, determined between wavelengths of 251 nm The residue from solution Sz weighs a maximum of 3 mg,
and 360 nm on solution S2' Use the reference solution as allowing for the blank test.
the compensation liquido
Oxidisable substances Immediately after preparation of Packaging
solution S2 (see 3.2.3), transfer to a borosilicate-glass fiask a See Sterile plastic containers jor human blood and blood
quantity corresponding to 8 per cent of the nominal capacity componems (3.2.3).
of the container. At the same time, prepare a blank using an
Labelling
equal volume of the freshly prepared reference solution in
another borosilicate-glass fiask. To each solution add See Sterile plastic containers jor human blood and blood
20.0 mL of 0.002 M potassium permanganate and 1 mL of components (3.2.3).
dilute sulfuric acid R. Allow to stand protected from light for
3. Sterile Containers of Plasticised Poly(Vinyl
15 mino To each sdlution add 0.1 g of potassium iodide R.
Chloride) for Human Blood Containing
Allow to Stand protected from light for 5 min and ti trate
Anticoagulant Solution
immediately with 0.01 M sodium thiosulfate, using 0.25 mL of
starch solution R as indicator. The difference between the 2 (Ph. Eur. method 3.2.5)
titrations is not more than 2.0 mL. Sterile plastic containers containing an anticoagulant solution
Extractable di(2-ethylhexyl) phthalate Extraction complying with the monograph Anticoagulant and preservative
solvent, ethanol (96 per cent) R diluted with water R to have a solutions jor human blood (0209) are used for the collection,
relative density (2.2.5) of 0.9389 to 0.9395, measured with a storage and administration of blood. Before filling they
pycnometer. comply with the description and characters given under
Empty sterile containers oj plasticised poly(vinyl chloride) jor
Stock solution Dissolve 0.100 g of di(2-ethylhexyl)
human blood and blood components (3.2.4).
phthalate R in the extraction solvent and dilute to 100.0 mL
with the same solvent. Unless otherwise authorised as described under Sterile plastic
containers jor human blood and blood components (3.2.3), the
Standard solutions. Into 5 separate 100 mL volumetric
nature and composition of the material from which the
fiasks, introduce respectively 1.0 mL, 2.0 mL, 5.0 mL,
containers are made should comply with the requirements
10.0 mL and 20.0 mL of stock solution and dilute to
prescribed for Materials based on plasticised poly(vinyl chlO1"ide)
100.0 mL with' extraction solvent.
jor containers jor human blood and blood components and jor
Measure the absorbances (2.2.25) of the standard solutions containers jor aqueous solutions jor intravenous injusion (3.1.1).
at the absorption maximum at 272 nm, using the extraction
solvent as compensation liquid and plot a curve of Tests
absorbance against the concentration of di(2-ethylhexyl) They comply with the tests prescribed for Sterile plastic
phthalate. containers jor human blood and blood components (3.2.3) and
Extraction procedure. Using the donor tubing and the with the following tests to measure the volume of
needle or adaptor, fill the empty container with a volume anticoagulant solution and to detect extractable matter.
V -A540 Appendix XIX E 2014
Volume of anticoagulant solution Empty the container, Tbe closures are compatible with the preparation for whicb
collecting the anticoagulant solution in a graduated cylinder. they are used tbrougbout Íts period of validity.
The volume does not differ by more than ± 10 per cent The manufacturer of the preparation must obtain from the
from the stated volume. supplier an assurance that the composition of the closure
Spectrophotometric examination (2.2.25) Measure the does not vary and that it is identical to that of tbe closure
absorbance of the anticoagulant solution from tbe container used during compatibility testing. When the supplier informs
between 250 nm and 350 nm, using as tbe compensation the manufacturer of the preparation of changes in the
liquid an anticoagulant solution of the same composition tbat composition, compatibility testing must be repeated, totally
has not been in contact with a plastic material. or partly, depending on the nature of the cbanges .
The absorbance at the maximum at 280 nm is not greater The closures are wasbed and may be sterilised before use.
than 0.5.
Extractable di(2-ethyIhexyl) phtbalate Carefully remove Characters
the anticoagulant solution by means of the flexible transfer Rubber closures are elastic; they are translucent or opaque
tube. Using a funnel fiued ro the tube, completely fill the and have no cbaracteristic colour, the larter depending on the
container with water R, leave in contact for 1 min squeezing additives used. Tbey are practically insoluble in
the container gently, then empty completely. Repeat the tetrahydrofuran, in whicb, however, a considerable reversible
rinsing. swelling may occur. They are bomogeneous and practically
Tbe container, so emptied and rinsed, complies with tbe test free froro flash and adventitious materials (for example fibres,
for extractable di(2-ethylhexyl) pbthalate prescribed for foreign particles, waste rubber).
Empty sterile plastic containers of plasticised poly(vinyl chloride) Identijication of the type of rubber used for the closures is not
for human blood and blood components (3.2.4). within the scope of this specijication. The identijication test given
below distinguishes elastomer and non-elaslOmer closures but does
Packaging and labelling not differentiate the various types of rubber. Other identity tests
See Sterile plastic containers for human blood and blood may be carried out with the aim of detecting differences in a batch
components (3.2.3). compared lO the e/osures used for compatibility testing. One or
more of the following analytical methods may be applied for this
purpose:
E. Rubber Closures for Containers for determination of relative density, determination of sulfated ash,
determination of sulfur content, thin-layer chromalOgraphy carried
Aqueous Parenteral Preparations out on an extract, ultraviolet absorption spectropholOmetry of an
(Ph. Eur. text 3.2.9, entitled Rubber closures for containers for extract, infrared absorption spectropholOmetry of a pyrolysate.
aqueous parenteral preparations for powders and for freeze-dn'ed
powders.) Identification
Rubber closures for containers for aqueous parenteral A. Tbe elasticity is sucb tbat a strip of material witb a
preparations for powders and for freeze-dried powders are cross-section of 1 mm2 to 5 mm 2 can be stretcbed by
made of materials obtained by vulcanisation (cross-linking) of bimd to at least twice its original lengtb. Having been
macromolecular organic substances (elastomers), with stretcbed to twice its length for 1 min, it contracts to less
appropriate additives. The specification also applies to than 1.2 times its originallength within 30 s.
closures for containers for powders and freeze-dried products B. Heat 1 g ro 2 g in a beat-resistant test-tube over an open
to be dissolved in water immediately before use. flame to dry the sample and continue beating until
Tbe specification does not apply ro closures made from pyrolysate vapours are condensed near the rop edge of
silicone elastomer (whicb are deaIt with in 3.1.9. Silicone the test-tube. Deposit a few drops of tbe pyrolysate on a
elastomer for c{osures and tubing), to laminated closures or to potassium bromide disc and examine by infrared
lacquered closures. The elastomers are produced from absorption spectrophotometry (2.2.24), comparing witb
natural or synthetic substances by polymerisation, the spectrum obtained with tbe type sample.
polyaddition or polycondensation. Tbe nature of the C. Tbe total asb (2.4.16) is within ± 10 per cent of the
principal components and of the various additives (for result obtained with tbe type sample.
example vulcanisers, accelerators, stabilisers, pigments)
depends on the properties required for the finisbed article. Tests
Rubber closures may be classified in 2 types: type I closures The samples 10 be analysed may be washed and sterilised before
are those wbicb meet the strictest requirements and which use.
are to be preferred; type II closures are tbose wbicb, baving
Solution S Introduce a number of uncut closures
mechanical properties suitable for special uses (for example,
corresponding to a surface area of about 100 cm 2 in a
multiple piercing), ca=ot meet requirements as severe as
suitable glass container, cover witb water for injections R, boil
those for tbe first category because of tbeir cbemical
for 5 min and rinse 5 times with cold water for injections R.
compositioq.
Place the wasi:led closures in a wide-necked flask (glass type
The closures cbosen for use with a particular preparation are 1, 3.2.1), add 200 mL of water for injections R and weigb.
such that: Cover the mouth of tbe flask with a borosilicate-glass beaker.
- the components of the preparation in contact with the Heat in an autoclave so that a temperature of 121 ± 2 oC is
closure are not adsorbed onto the surface of the closure reached within 20 min to 30 min and maintain at this
and do not migrate into or througb the closure ro an temperature for 30 mino Cool to room temperature over
extent sufficient ro affect the preparation adversely, about 30 mino Make up ro tbe original mas s witb water for
- the closure does not yield to the preparation substances in injections R. Shake and immediately separate the solution
quantities sufficient ro affect its stability or ro present a from the rubber by decantation . Shake solution S before
risk of toxicity. each test
2014 Appendix XIX F V-A541
Blank Prepare a blank in the same manner using 200 mL For the tests for penetrability, fragmentation and self-sealing, use
of water for injections R. the closures treated as described for the preparation of solution S
Appearance of solution Solution S is not more and allowed ro dry.
opalescent than reference suspension II for type I closures Penetrability For closures intended 10 be pierced by a
and is not more opalescent than reference suspension III for hypodermic needle, carry out the following test. Fill 10
type II closures (2.2.1). Solution S is not more intensely suitable vials to the nominal volume with water R, fit the
coloured than reference solution GYs (2.2.2, Method 1l) . closures to be examined and secure with a cap. Using for
Acidity or aIkalinity To 20 mL of solution S add 0.1 mL each closure a new, lubricated long-bevel(l) (bevel angle
of bromothymol blue solution R1. Not more than 0.3 mL of 12 ± 2°) hypodermic needle with an external diameter of
0. 01 M sodium hydroxide or 0.8 mL of 0.01 M hydrochloric 0.8 mm, pierce the closures with the needle perpendicular 10
acid is required to obtain either a blue or a yellow colour, the surface. The force required for piercing, determined with
respectively. an accuracy of ± 0.25 N (25 gí), is not greater than 10 N
(1 kgí) for each closure.
Absorbance Carry out the test within 5 h of preparation of
solution S. Filter solution S on a membrane filter having Fragmentation For closures intended to be pierced by a
approximately 0.45 Jlm pores rejecting the first few millilitres hypodermic needle, carry out the following test. If the
of filtrate. Measure the absorbance (2.2.25) of the filtrate at closures are to be used for aqueous preparations, place in 12
wavelengths from 220 nm to 360 nm using the blank (see clean vials a volume of water R corresponding to the nominal
solution S) as compensation liquido At these wavelengths, the volume minus 4 mL, close the vials with the closures to be
absorbance do es not exceed 0.2 for type I closures or 4.0 for examined, secure with a cap and allow 10 stand for 16 h.
type II closures. If necessary, dilute the filtrate before If the closures are 10 be used with dry preparations, close 12
measurement of the absorbance and correct the result for the clean vials with the closures to be examined. Using a
dilution. lubricated long-bevel(2) (bevel angle 12 ± 2°) hypodermic
needle with an external diameter of 0.8 mm fitted to a clean
Reducing substances Carry out the test within 4 h of
preparation of solution S. To 20.0 mL of solution S add I mL
syringe, inject into the vial 1 mL of water R and remove
of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium 1 mL of air; carry out this operation 4 times for each
closure, piercing each time at a different site. Use a new
permanganate. Boil for 3 min. Coo!. Add 1 g of potassium
needle for each closure and check that the needle is not
iodide R and titrate irnmediately with 0.01 M sodium
blunted during the test. Pass the liquid in the vials through a
thiosulfate, using 0.25 mL of starch solution R as indicator.
Carry out a titration using 20.0 mL of the blank. filter having approximately 0.5 ~lm pores. Count the
fragments of rubber visible 10 the naked eye. The total
The difference between the titration volumes is not greater
number of fragments does not exceed 5. This limit is based
than 3.0 mL for type I closures and 7.0 mL for type II
on the assumption that fragments with a diameter equal 10
closures.
or greater than 50 Jlm are visible ro the naked eye; in cases
Ammonium (2.4.1) Maximum 2 ppm. of doubt or dispute, the fragments are examined with a
Dilute 5 mL of solution S to 14 mL with water R . microscope to verify their nature and size.
The solution complies with limit test A. Se1f-sealing test For closures intended to be used with
Extractable zinc Maximum of 5 Jlg of extractable Zn per multidose containers, carry out the following test. Fill 10
millilitre of solution S. suitable vials 10 the nominal volume with water R, fit the
Atomic absorption spectrometry (2.2.23, Method l) . closures to be examined and secure with a cap. Using for
Test solution Dilute 10.0 mL of solution S to lOO mL each closure a new hypodermic needle with an external
with O. 1 M hydrochloric acid. diameter of 0.8 mm, pierce each closure 10 times, piercing
each time at a different site. Immerse the vials upright in a
1 gIL solution of methylene blue R and reduce the external
zinc standard solution (10 ppm Zn) R diluted with 0.1 M
hydrochloric acid. .
pressure by 27 kPa for 10 mino Restore atmospheric pressure
and leave the vials immersed for 30 mino Rinse the outside
Source Zinc hollow-cathode lampo of the vials. None of the vials contains any trace of coloured
Wavelength 213.9 nm. solution.
Flame Air-acetylene.
Extractable heavy metals (2.4.8) Maximum 2 ppm.
Solution S complies with limit test A. Prepare the standard F. Sets for the Transfusion of Blood and
using lead standard solution (2 ppm Pb) R. Blood Components
Residue on evaporation Evaporaté 50.0 mL of solution S
to dryness on a water-bath and dry at 100 oC to 105 oc. (Ph. Eur. method 3.2.6)
The residue weighs not more than 2.0 mg for type I rubber Sets for the transfusion of blood and blood components
and not more than 4.0 mg for type II rubber. consist principally of plastic tubing to which are fitted the
parts necessary ro enable the set to be used for transfusion in
Volatile sulfides Place closures, cut if necessary, with a
the appropriate manner. Sets include a closure-piercing
total surface area of 20 ± 2 cm 2 in a 100 mL conical ftask
device, a blood filter, a drip chamber, a ftow regulator, a
and add 50 mL of a 20 giL solution of citric acid R. Place a
Luer connector and, usually, a site that allows an injection 10
pie ce of lead acetate paper R over the mouth of the ftask and
be made at the time of use. When the sets are to be used
maintain the paper in position by placing over it an inverted
with containers requiring an air-filter, this may be
weighing bottle. Reat in an autoclave at 121 ± 2 oC for
incorporated in the closure-piercing device or a separate air-
30 mino Any black stain on the paper is not more intense
inler device may be used. The chamber enclosing the blood
than that of a standard prepared at the same time in the
same manner using 0.154 mg of sodium sulfide R and 50 mL
1 See ISO 7864 "Ste/ile hypodmnic needles for single use".
of a 20 gIL solution of citric acid R.
2 See ISO 7864 "Sterile hypodel'lnic needles for single use".
V -A542 Appendix XIX F 2014
filter, the drip chamber and the main tubing are transparento Verify the absence of peaks interfering with the ethylene
The materials chosen and the design of the set are such as ro oxide peak by carrying out the test using an unsterilised set
ensure absence of haemolytic effects. The sets comply with or using the same chromatographic system with the following
currem standards regarding dimensions and performance. modifications.
All parts of the set that may be in comact with blood and Column
blood components are sterile and pyrogen-free. Each set is - size: l = 3 m, 0 = 3.2 mm;
presemed in an individual package that maimains the sterility - stationary phase: silanised diatomaceous earth for gas
of the comems. The sets are not ro be re-sterilised or chromatography R impregnated with
re-used. triscyanoethoxypropane R (2 g per 10 g);
Sets for the transfusion of blood and blood componems are - tempera tu re: 60 oc.
manufacrured in accordance with the rules of good
Ethylene oxide solution Prepare under a ventilated hood.
manufacruring practice for medical devices and any relevam
Place 50.0 mL of dimethylacetamide R in a 50 mL vial,
national regulations.
stopper, secure the stopper and weigh to the nearest 0.1 mg.
Tests Fill a 50 mL polyethylene or polypropylene syringe with
gaseous ethylene oxide R, allow the gas to remain in contact
Carry out the tests on sterilised sets. with the syringe for about 3 min, empty the syringe and fill
Solution S Make a cIosed circulation system from 3 sets again with 50 mL of gaseous ethylene oxide R. Fit a
and a 300 mL borosilicate-glass vessel. Fit to the vessel a hypodermic needle ro the syringe and reduce the volume of
suitable thermostat device that maintains the temperarure of gas in the syringe from 50 mL to 25 mL. Inject these 25 mL
the liquid in the vessel at 37 ± 1 °C. Circulate 250 mL of of ethylene oxide slowly imo the vial, shaking gemly and
water for injections R through the system in the direction used avoiding comact between the needle and the liquido Weigh
for transfusion for 2 h at arate of 1 IJh (for example using a the vial again: the increase in mass is 45 mg to 60 mg and is
peristaltic pump applied ro as short a piece of suitable used to calculate the exact concentration of the solution
silicone elastomer tubing as possible). Collect the whole of (about 1 gIL) .
the solution and allow to cool.
Test Weigh the set after removing the package. Cut the set
Appearance of solution Solution S is cIear (2.2.1) and imo pieces of maximum dimension 1 cm and place the
colourless (2.2.2, Method IJ). pieces in a 250-500 mL vial comaining 150 mL of
Acidity or aIkalinity To 25 mL of solution S add dimethylacetamide R. Close the vial with a suitable sropper
0.15 mL of BRP indicator solution R. Not more than 0.5 mL and secure the sropper. Place the vial in an oven at
of o. 01 M sodium hydroxide is required to change the colour 70 ± 1 oC for 16 h. Remove 1 mL of the hot gas from the
of the indicator ro blue. To 25 mL of solution S add 0.2 mL vial and inject it onto the column. From the calibration
of methyl orange solution R. Not more than 0.5 mL of 0.01 M curve and the height of the peak obtained, calculate the mas s
hydrochloric acid is required ro reach the beginning of the of ethylene oxide in the vial.
colour change of the indicaror. Calibration curve In a series of 7 vials of the same type
Absorbance (2.2.25) Maxirnum 0.30, determined between as that used for the test and each comaining 150 mL of
wavelengths of 230 nm and 250 nm on solution S; dimethylacetamide R, place respectively O mL, 0.05 mL,
maximum 0.15, determined between wavelengths of 251 nm 0.10 mL, 0.20 mL, 0.50 mL, 1.00 mL and 2.00 mL ofthe
and 360 nm on solution S. ethylene oxide solution, i.e. about O ¡.tg, 50 ¡.tg, 100 ¡.tg,
Reducing substances Carry out the test within 4 h of 200 ¡.tg, 500 ¡.tg, 1000 ¡.tg and 2000 ¡.tg of ethylene oxide.
preparation of solution S. To 20.0 mL of solution S add 1 mL Stopper the vials, secure the stoppers and place the vials in
of dilute sulfuric acid R and 20.0 mL of 0.002 M potassium an oven at 70 ± 1 oC for 16 h. Inject 1 mL of the hot gas
permanganate. Boil for 3 min and cool immediately. Add 1 g from each vial onto the column and draw a calibration curve
of potassium iodide R and titrate with O. 01 M sodium from the heights of the peaks and the mass of ethylene oxide
thiosulfate ·using 0.25 mL of starch solution R as indicator. in each flask.
Carry out a blank test using 20 mL of water for injections R. Limit If the label sta tes that ethylene oxide has been used
The difference between the titration volumes is not greater for sterilisation:
than 2.0 mL. - ethylene oxide: maximum 10 ppm.
Ethylene oxide Gas chromatography (2.2.28) . Extraneous particles FilI the set via the normal inlet with
Column a 0.1 gIL solution of sodium laurilsulfate R, previously filtered
- material: stainless steel; through a simered-glass filter (16) (2.1.2) and heated to
37 oC. Collect the liquid via the normal oudet. When
- size: l = 1.5 m, 0 = 6.4 mm;
examined under suitable conditions of visibility, the liquid is
- stationary phase: silanised diatomaceous earth for gas cIear and practically free from visible particIes and filamems
chromatography R impregnated with macrogol 1500 R (3 g
(it is assumed that particIes and filamems with a diameter
per 10 g).
equal to or greater than 50 ¡.tm are visible to the naked eye).
Flow rate Pass through a complete set with the flow
Flow rate 20 mUmin. regulator fuIly open 50 mL of a solution having a viscosity of
Temperature 3 mPa·s (3 cP) (for example a 33 gIL solution of macrogol
- column: 40 oC; 4000 R at 20 oC) under a static head of 1 m. The time
- injection port: 100 oC; required for passage of 50 mL of the solution is not greater
than 90 s.
- detector: 150 oC .
Resistance to pressure Make tight the extremities of the
Detection Flame ionisation. set and any air-inlet device. Connect the set ro a compressed
air outIet fined with a pressure regulator. Immerse the set in
2014 Appendix XIX G V -A543
a tank of water at 20-23 oC. Apply progressively an excess Silicone oil (3.1.8) may be applied 10 the intemal wall of the
pressure of 100 kPa and maintain for 1 mino No air bubble barrel to assist in the smooth operation of the syringe but
escapes from the seto there remains no excess capable of contaminating the
Transparency Use as reference suspension the primary contents at the time of use. The inks, glues and adhesives for
opalescent suspension (2.2.1) diluted 1 in 8 for sets having the marking on the syringe or on the package and, where
tubing with an external diameter less than 5 mm and diluted necessary, the assembly of the syringe and its package, do not
1 in 16 for sets having tubing with an external diameter of migrate across the walls.
5 mm or greater. Circulate the reference suspension through
the set and compare with a set from the same batch filled Tests
with water R . The opalescence and presence of bubbles are Solution S Prepare the solution in a manner that avoids
discernible. contamination by foreign partic1es. Using a sufficient number
Residue on evaporation Evaporate 50.0 mL of solution S of syringes 10 produce 50 mL of solution, fill the syringes 10
to dryness on a water-bath and dry to constant mass in an their nominal volume with water lor injections R and maintain
oven at 100-105 oc. Carry out a blank test using 50.0 mL of at 37 oC for 24 h . Combine the contents of the syringes in a
water lor injections R. The difference between the mas ses of suitable borosilicate-glass container.
the residues is not greater than 1.5 mg. Appearance of solution Solution S is c1ear (2.2. 1) and
Sterility (2.6.1) The sets comply with the test for sterility. colourless (2.2.2, Method JI) and is practically free from
If the sets are stated to be sterile only internally, pass 50 mL foreign solid partic1es.
of buffered sodium chloride-peptone solution pH 7.0 Acidity or alkalinity To 20 mL of solution S add 0.1 mL
(2.6.12) through the set and use to carry out the test by the of bromothymol blue solution Rl. Not more than 0.3 mL of
membrane filtration method. O. 01 M sodium hydroxide or O. O1 M hydrochlon'c acid is
If the sets are stated to be sterile both internally and required 10 change the colour of the indicator.
externally, open the package with the necessary aseptic Absorbance (2.2.25) Maximum 0.40, determined between
precautions and: wavelengths of 220 nm and 360 nm on solution S.
- for the direct inoculation method, place the set or its Ethylene oxide Gas chromatography (2.2.28).
components in a suitable container containing a sufficient Column
quantity of the culture medium to ensure complete - material: stainless steel;
immersion;
- size: 1 = 1.5 m, 0 = 6.4 mm;
- for the membrane filtration method, place the set or its
- slationary phase: silanised diatomaceous earth lor gas
components in a suitable container containing a sufficient
chromatography R impregnated with macrogol 1500 R (3 g
quantity of buffered sodium chloride-peptone solution
per 10 g) .
pH 7.0 (2.6. 12) to allow total rinsing for 10 mino
Camer gas helium lor chromatography R .
Pyrogens (2.6.8) Connect together 5 sets and pass
through the assembly at a fiow rate not exceeding Flow rate: 20 mUmin.
10 mUmin 250 mL of a sterile, pyrogen-free 9 gIL solution Temperature
of sodium chloride R. Collect the solution aseptically in a - Column: 40 oC;
pyrogen-free container. The solution complies with the test - Jnjection port: 100 oC;
for pyrogens . rnject per kilogram of the rabbit's mass, 10 mL
- D etector: 150 oc.
of the solution.
Labelling Verify the absence of peaks interfering with the ethylene
The label states, where applicable, that the set has been oxide peak, either by carrying out the test using an
sterilised using ethylene oxide. unsterilised syringe or using the same chromatographic
system with the following modifications:
Column
G. Sterile Single-use Plastic Syringes - size: 1 = 3 m, 0 = 3.2 mm;
- stationary phase: silanised diatomaceous earth lor gas
(Ph. Éur. method 3.2.8)
chromatography R impregnated with
Sterile single-use plastic syringes are medical devices intended
triscyanoethoxypropane R (2 g per 10 g);
for irnmediate use for the administration of injectable
preparations. They are supplied sterile and pyrogen-free and - temperature: 60 oc.
are not to be re-sterilised or re-used. They consist of a Ethylene oxide solution Prepare under a ventilated hood.
syringe barrel and a piston which may have an elastomer Place 50.0 mL of dimethylacetamide R in a 50 mL vial,
sealing ring; they may be fitted with a needle which may be stopper, secure the stopper and weigh to the nearest 0.1 mg.
non-detachable. Each syringe is presented with individual Fill a 50 mL polyethylene or polypropylene syringe with
protection for maintaining sterility. gaseous ethylene oxide R, allow the gas to remain in contact
The barrel of the syringe is sufficiently transparent to permit with the syringe for about 3 min, empty the syringe and fill
dosages to be read without difficulty and allow air bubbles again with 50 mL of gaseous ethylene oxide R. Fit a
and foreign partic1es to be discemed. hypodermic needle 10 the syringe and reduce the volume of
gas in the syringe from 50 mL to 25 mL. rnject these 25 mL
The plastics and elastomer material s of which the barrel and
of ethylene oxide slowly into the vial, shaking gently and
piston are made comply with the appropriate specification or
avoiding contact between the needle and the liquido Weigh
with the requirements of the competent authority. The most
the vial again: the increase in mas s is 45 mg 10 60 mg and is
commonly used materials are polypropylene and
used to calculate the exact concentration of the solution
polyethylene. The syringes comply with current standards
(about 1 gIL).
regarding dimensions and performance.
V -A544 Appendix XIX G 2014
Calibration curve In a series of seven vials of the same components and place each in a suitable container
type as that used for the test and each containing 150 mL of containing sufficient culture media to cover the part
dimethylacetamide R, place respectively O mL, 0.05 mL, completely. Use both the recommended media (2.6.1).
0.10 mL, 0.20 mL, 0.50 mL, 1.00 mL and 2.00 mL ofthe Syringes stated to be sterile only internally comply with the test for
ethylene oxide solution, i.e . about O ¡.¡g, 50 ¡.¡g, 100 ~lg, sterility camed out as follows. Use 50 mL of inoculation
200 ¡.¡g, 500 ¡.¡g, 1000 ¡.¡g and 2000 ¡.¡g of ethylene oxide. medium for each test syringe. Using aseptic technique,
Stopper the vials, secure the stoppers and place the vials in remove the needle protector and submerge the needle in the
an oven at 70 ± 1 oC for 16 h. Inject 1 mL of the hot gas culture medium. Flush the syringe five times by withdrawing
from each vial onto the column and draw a calibration curve the plunger to its fullest extent.
from the heights of the peaks and the mass of ethylene oxide Pyrogens (2.6.8) Syringes with a nominal volume equal to
in each fiask. or greater than 15 mL comply with the test for pyrogens. Fill
Test Weigh the syringe after removing the package. a minimum of three syringes to their nominal volume with a
Cut the syringe into pieces of maximum dimension 1 cm pyrogen-free 9 gIL solution of sodium ehloride R and maintain
and place the pieces in a 250 mL to 500 mL vial containing at a temperature of 37 oC for 2 h. Combine the solutions
150 mL of dimethylacetamide R. Close the vial with a suitable aseptically in a pyrogen-free container and carry out the test
stopper and secure the stopper. Place the vial in an oven at immediately. Inject per kilogram of the rabbit's mass 10 mL
70 ± 1 oC for 16 h. Remove 1 mL of the hot gas from the of the solution.
vial and inject it onto the column. From the calibration
curve and the height of the peak obtained, calculate the mas s Labelling
of ethylene oxide in the vial. The label on the package sta tes:
Limit: If the label states that ethylene oxide has been used - the batch number;
for sterilisation: - a description of the syringe;
- ethylene oxide: maxirnum 10 ppm. - that the syringe is for single-use only.
Silicone oi! Calculate the internal surface are a of a syringe The label on the outer package states:
in square centimetres using the following expression:
- the method of sterilisation;
- that the syringe is sterile or that it is sterile only internally;
- the identity of the manufacturer;
- that the syringe is not to be used if the packaging is
v = nominal volume of the syringe, in cubic centimetres, damaged or the sterility protector is loose.
h = height of the graduation, in centimetres.
Take a sufficient number of syringes to give an internal
surface area of 100 cm2 to 200 cm 2 . Aspirate into each
syringe a volume of methylene chloride R equal to half the
nominal volume and make up to the nominal volume with
airo Rinse the internal surface corresponding to the nominal
volume with the solvent by inverting the syringe ten times in
succession with the needle fitting c10sed by a finger covered
by a plastic film inert to methylene chloride. Expel the
extracts into a tared dish and repeat the operation. Evaporate
the combined extracts to dryness on a water-bath. Dry at
100-105 oC for 1 h. The residue weighs not more than
0.25 mg per square centimetre of internal surface area.
Examine the residue by infrared absorption
spectrophotometry (2.2.24) . It shows absorption bands
typical of silicone oil at 805 cm - 1, 1020 cm - 1, 1095 cm - I,
1260 Crp - I and 2960 cm- l.
Reducing substances To 20.0 mL of solution S add
2 mL of sulfuric acid R and 20.0 mL of 0.002 M potassium
permanganate. Boil for 3 min. Cool immediately. Add 1 g of
potassium iodide R and titrate immediately with 0.01 M
sodium thiosulfate using 0.25 mL of starch solution R as
indicator. C~rry out a blank titration using 20.0 mL of water
for injections R. The difference between the titration volumes
is not greater than 3.0 mL.
Transparency Fill a syringe with water R (blank) and fill
another with a 1 in 10 dilution of primary opalescent
suspension (2.2.1). Use primary opalescent suspension that
has been allowed to stand at 20 ± 2 oC for 24 h before use.
Compare with the naked eye in diffused light against a dark
background. The opalescence of the suspension is detectable
when compared with the blank.
Sterility (2. 6.1) Sylinges srated to be ste1'11e comply with the
test for sterility cam'ed out as follows. Using aseptic technique,
open the package, withdraw the syringe, separate the
2014 Appendix XX A V -A545
Internal standard solution Using a microsyringe, inject

Appendix XX 10 ¡.¡L of ether R into 20.0 mL of dimethylaeetamide R,
immersing the tip of the needle in the solvent. Immediately
before use, dilute the solution to 1000 times its volume with
Materials Used for the Manufacture of dimethylaeetamide R.
Containers Test solution Place 1.000 g of the material to be
examined in a 50 mL vial and add 10.0 mL of the internal
(Ph. Eur. text 3.1)
standard solution. Close the vial and secure the stopper.
The materials described in this chapter are used for the
Shake, avoiding contact between the stopper and the liquido
manufacture of containers for pharmaceutical use. Their use
Place the vial in a water-bath at 60 ± 1 oC for 2 h.
may also be considered for the manufacture of part or aH of
objects used for medico-surgical purposes. Vinyl chloride primary solution Prepare under a
ventilated hood. Place 50.0 mL of dimethylaeetamide R in a
Materials and polymers other than those described in the
50 mL vial, stopper the vial, secure the stopper and weigh to
Pharmacopoeia may be used subject to approval in each case
the nearest 0.1 mg. Fill a 50 mL polyethylene or
by the competent authoriry responsible for the licensing for
polypropylene syringe with gaseous vinyl ehloride R, allow the
sale of the preparation in the container.
gas to remain in contact with the syringe for about 3 min,
Transmissible spongiforrn encephalopathies (5.2.8) A empry the syringe and fill again with 50 roL of gaseous vinyl
risk assessment of the product with respect to transmissible ehloride R. Fit a hypodermic needle to the syringe and reduce
spongiform encephalopathies is carried out, and suitable the voluroe of gas in the syringe from 50 mL to 25 mL.
measures are taken to minimise any such risk. Inject the remaining 25 mL of vinyl chloride slowly into the
vial shaking gently and avoiding contact between the liquid
and the needle. Weigh the vial again; the increase in mas s is
A. Materials for Containers for Human about 60 mg (1 ¡.¡L of the solution thus obtained contains
about 1.2 ¡.¡g ofvinyl chloride). AHow to stand for 2 hours.
Blood and Blood Components Keep the primary solution in a refrigerator.
(Ph. Eur. method 3.1.1) Vinyl chloride standard solution Vinyl chloride primary
NOTE: jor materials based on plastieised poly(vinyl ehloride) jor solution, dimethylaeetamide R (1:3 V/V).
containers jor aqueous solutions jor intravenous injusion, see text Reference solutions Place 10.0 mL of the intemal
3.1.14. standard solution in each of six 50 mL vials. Close the vials
Plastic containers for the collection, storage, processing and and secure the stoppers. Inject 1 ¡.¡L, 2 ¡.¡L, 3 ~lL, 5 ~lL and
administration of blood and its components may be 10 ¡.¡L, respectively, of the vinyl chloride standard solution
manufactured fro m one or more polymers, if necessary with into five of the vials. The six solutions thus obtained contain,
certain additives. respectively, O ¡.¡g, about 0.3 ¡.¡g, 0.6 ¡.¡g, 0.9 ¡.¡g, 1.5 ¡.¡g and
If all or part of the container consists of a material described 3 ¡.¡g of vinyl chloride. Shake, avoiding contact between the
in a text of the Pharmacopoeia, the qualiry of the material is stopper and the liquido Place the vials in a water-bath at
controlled by the methods indicated in that texto (See 60 ± 1 oC for 2 h .
3.1.1.1. Materials based on plastieised poly(vinyl ehloride) jor Column:
eontainers jor human blood and blood eonlponents). - material: stainless steel;
In normal conditions of use the materials and containers - size: I = 3 m, 0 = 3 mm;
made from such materials do not release monomers, or other
- stationary phase: silanised diatomaeeous earth jor gas
substances, in amounts likely to be harmful nor do they lead
ehromatography R impregnated with 5 per cent mhn of
to any abnormal modifications of the blood or blood
dimethylstearylamide R and 5 per cent m/m of maerogol
components.
400R.
1. Materials Based on Plasticised Poly(Vinyl Carrier gas nitrogen jor ehromatography R.
Ch1oride) for Containers for Human Blood and Flow rate 30 mUmin.
Blood Components
Temperature:
(Ph. Eur. method 3.1.1.1) - eolumn: 45 oC;
Definition - injeetion port: 100 oC;
Materials based on plasticised poly(vinyl chloride) contain - detector: 150 oC.
not less than 55 per cent of poly(vinyl chloride) and contain
various additives, in addition to the high-molecular-mass
polymer obtained by polymerisatiort ofvinyl chloride. Injection 1 mL of the headspace.
Materials based on plasticised poly(vinyl chloride) for Limit:
containers for human blood and blood components are - vinyl chloride: maximum 1 ppm.
defined by the nature and the proportions of the substances ADDITIVES
used in their manufacture . A certain number of additives are added to the polymers to
Production optimise their chemical, physical and mechanical properties
Materials based on plasticised poly(vinyl chloride) are in order to adapt them for the intended use. AH these
produced by polymerisation methods that guarantee a additives are chosen from the following list which specifies
residual vinyl chloride content of less than 1 ppm. for each product the maximum aHowable content:
The manufacturing process is validated to demonstrate that - di(2-ethylhexyl)phthalate (plastic additive 01) : maximum
the product complies with the following test. 40 per cent;
Vinyl chloride Head-space gas chromatography (2.2.28).
V-A546 Appendix XX A 2014
- zinc octanoate (zinc 2-ethylhexanoate) (plastic additive Tests

02): maximum 1 per cent; 1f necessary, before use, cut the samples of the material to be
- calcium stearate or zinc stearate: maximum 1 per cent or examined into pieces of maximum dimension on a side of not
1 per cent of a mixture of the two; greater than 1 cm.
- N,N'-diacylethylenediamines (plastic additive 03): Solution SI Place 5.0 g of the material to be examined in
maximum 1 per cent; a combustion fiask. Add 30 mL of suljuric acid R and heat
- one of the following epoxidised oils: maximum until a black, syrupy mass is obtained. Cool and add
10 per cent or 10 per cent of a mixture of the two: carefully 10 mL of strong hydrogen peroxide solution R. Reat
gently. AlIow to cool and add 1 mL of strong hydrogen
- epoxidised soya oil (plastic additive 04), of which the
peroxide solution R; repeat by alternating evaporation and
oxiran oxygen content is 6 per cent to 8 per cent and
addition of hydrogen peroxide solution until a colourless
the iodine value is not greater than 6;
liquid is obtained. Reduce the volume to about 10 mL. Cool
- epoxidised linseed oil (plastic additive 05), of which and dilute to 50.0 mL with water R.
the oxiran oxygen content is not greater than
Solution S2 Place 25 g of the material to be examined in
10 per cent and the iodine value is not greater than 7.
a borosilicate-glass fiask. Add 500 mL of water for injections R
Very low amounts of antioxidants added to the vinyl chloride and cover the neck of the fiask with a borosilicate-glass
monomer may be detected in the polymer. beaker. Reat in an autoclave at 121 ± 2 oC for 20 mino
No antioxidant additive may be added to the polymer. Allow to cool and decant the solution. Make the volume up
Ultramarine blue is the only colouring material permined to to 500 mL.
be added. Appearance of solution S2 Solution S2 is clear (2.2.1)
The supplier of the material must be able to demonstrate and colourless (2.2.2, Method 11).
that the qualitative and quantitative composition of the type Acidity or aIkalinity To 100 mL of solution S2, add
sample is satisfactory for each production batch. 0.15 mL of BRP indicator solution R . Not more than 1.5 mL
Characters of 0.01 M sodium hydroxide is required to change the colour
of the indicator to blue. To 100 mL of solution S2 add
Colourless or pale yellow powder, beads, granules or, after
0.2 mL of methyl orange solution R. Not more than 1.0 mL of
transformation, translucent sheets of varying thickness or
0.01 M hydrochloric acid is required to initiate the colour
containers, with a slight odour. On combustion it gives off
change of the indicator from yellow to orange.
dense, black smoke.
Absorbance (2.2.25) Evaporate 100.0 mL of solution
Identification S2 to dryness. Dissolve the residue in 5.0 mL of hexane R.
1f necessary, before use, cut the samples of the material to be From 250 nm to 310 nm the absorbance is not greater than
examined into pieces of maximum dimension on a side of not 0.25.
greater than 1 cm.
To 2.0 g of the material to be examined add 200 mL of 'preparation of solution S2. To 20.0 mL of solution S2 add
peroxide-free ether R and heat under a refiux condenser for 1 mL of dilute sulfuric acid R and 20.0 mL of 0.002 M
8 h . Separate the residue B and the solution A by filtration. potassium permanganate. Boil under a refiux condenser for
Evaporate solution A to dryness under reduced pressure in a 3 min and cool immediately. Add 1 g of potassium iodide R
water-bath at 30 oc. Dissolve the residue in 10 mL of and titrate immediately with O. 01 M sodiUll1 thiosulfate, using
toluene R (solution Al) . Dissolve the residue B in 60 mL of 0.25 mL of starch solution R as indicator. Carry out a blank
ethylene chloride ·R, heating on a water-bath under a refiux titration using 20 mL of water for injections R. The difference
condenser. Filter. Add the solution obtained dropwise and between the two titration volumes is not more than 2.0 mL.
with vigorous shaking to 600 mL of heptane R heated almost Prirnary aromatic amines Maximum 20 ppm.
to boiling. Separate the coagulum B1 and the organic
To 2.5 mL of solution Al obtained during the identification,
solution by hot filtration. Allow the latter to cool; separate add 6 mL of water R and 4 mL of O. 1 M hydrochloric acid.
the precipitate B2 that forms and filter through a tared Shake vigorously and discard the upper layer. To the
sintered-glass filter (40) (2.1.2) .
aqueous layer add 0.4 mL of a freshly prepared 10 gIL
A. Infrared absorption spectrophotometry (2.2.24). solution of sodium nitrite R . Mix and allow to stand for
Preparation Dissolve the coagulum B 1 in 30 mL of 1 minoAdd 0.8 mL of a 25 gIL solution of ammonium
tetrahydrofuran R and add, in small volumes with shaking, sulfamate R, allow to stand for 1 min and add 2 mL of a
40 mL of anhydrous ethanol R. Separate the precipita te B3 by 5 giL solution of naphthylethylenediamine dihydrochloride R .
filtration and dry in vacuo at a temperature not exceeding After 30 min, any colour in the solution is not more intense
50 oC over diphosphorus pentoxide R. Dissolve a few than that in a standard prepared at the same time in the
milligrams of precipitate B3 in 1 mL of tetrahydrofuran R, same manner replacing the aqueous layer with a mixture of
place a few drops of the solution obtained on a sodium 1 mL of a 0.01 giL solution of naphthylamine R in 0.1 M
chloride plate and evaporate to dryness in an oven at hydrochloric acid, 5 mL of water R and 4 mL of 0.1 M
100-105 oC. hydrochloric acid instead of the aqueous layer.
Comparison poly(vinyl chloride) CRS. Plastic additives 01, 04 and 05 Thin-Iayer
B. Infrared absorption spectrophotometry (2.2.24). chromatography (2.2.27).
Examine the residue C obtained in the test for plastic Reference solutions Prepare 0.1 mglmL solutions of
additives 01, 04 and 05. plastic additive 01 CRS, plastic additive 04 CRS and plastic
additive 05 CRS, respectively, in toluene R.
Comparison plastic additive 01 CRS.
Plate TLC silica gel GF2 54 plate R.
Mobile phase toluene R.
2014 Appendix XX A V-A547
Application 0.5 mL of solurion Al obtained during the Test solution Use the test solution prepared for the
identification as a band 30 mm by 3 mm and 5 IlL of each determination of barium.
reference solution. Reference solution A solution containing 50.0 ppm of
Development Over a path of 15 cm. calcium prepared by dilution of calcium standard solution
Drying In airo (400 ppm Ca) R with 0.1 M hydrochloric acid.
Detection A Examine in ultraviolet light at 254 nm. Wavelength Use the emission of calcium at 315.89 nm,
the spectral background being taken at 315 .60 nm.
Locate the zone corresponding to plastic additive 01
(Rp = about 0.4) . Remove the are a of silica gel Verify the absence of calcium in the hydrochloric acid used.
corresponding to this zone and shake with 40 mL of ether R Tin Maximum 20 ppm.
for 1 mino Filter, rinse with two quantities, each of 10 mL of Inductively coupled plasma-atomic emission spectrometry
ether R, add the rinsings to the filtrate and evaporate to (2.2.57).
dryness. The residue C weighs not more than 40 mg.
Test solution Dilute solution SIlO times with water R
Detection B Expose the plate to iodine vapour for 5 mino immediately before use.
Examine the chromatogram and locate the band Reference solution Introduce 2 mL of tin standard solution
corresponding to plastic additives 04 and 05 (R p = O). (5 ppm Sn) R into a 50 mL flask containing 5 mL of a
Remove the area of silica gel corresponding to this zone. 20 per cent V/V solurion of suljuric acid R and dilute to
Similarly remove a corresponding area of silica gel as a blank 50 mL with water R immediately before use.
reference. Separately shake both samples for 15 min with
Wavelength Use the emission oftin at 189.99 nm, the
40 mL of methanol R . Filter, rinse with 2 quantities, each of
spectral background being taken at 190.10 nm.
10 mL of methanol R, add the rinsings to the filtrate and
evaporate to dryness. The difference between the mas ses of Verify the absence of tin in the sulfuric acid used.
both residues is not more than 10 mg. Zinc Maximum 0.2 per cent.
P1astic additive 03 Atomic absorption spectrometry (2.2.23, Method I).
Wash precipitate B2 obtained during the identification and Test solution Dilute solution SIl 00 times with 0.1 M
contained in the tared sintered-glass filter (40) (2.1.2) with hydrochloric acid.
anhydrous ethanol R. Dry to constant mass over diphosphorus Reference solutions Prepare the reference solutions using
pentoxide R and weigh the filter. The residue weighs not more zinc standard solution (100 ppm Zn) R, diluting with 0.1 M
than 20 mg. hydrochloric acid.
Infrared absorption spectrophotometry (2.2.24). Source zinc hollow-cathode lamp.
Preparation the residue obtained above. Wavelength 213.9 nm.
Comparison plastic additive 03 CRS. Atomisation device Air-acetylene flameo
Barium Maximum 5 ppm. Verify the absence of zinc in the hydrochloric acid used .
Inductively coupled plasma-atomic emission spectrometry Heavy metals (2. 4.8) Maximum 50 ppm.
(2.2.57) . To 10 mL of solution SI add 0.5 mL of phenolphthalein
Test solution Ignite 1.0 g of the substance to be examined solution R and then strong sodium hydroxide solution R until a
in a silica crucible. Take up the residue with 10 mL of pale pink colour is obtained. Dilute to 25 mL with water R .
hydrochloric acid R and evaporate to dryness on a water-bath. 12 mL of the solution complies with test A. Prepare the
Take up the residue with 20 mL of 0. 1 M hydrochloric acid. reference solution using lead standard solution (2 ppm Pb) R.
Reference solution A solurion containing 0.25 ppm of Water extractable substances Maximum 0.3 per cent.
barium prepared by dilution of barium standard solution Evaporate 50 mL of solution S2 to dryness on a water-bath
(50 ppm Ba) R with 0.1 M hydrochloric acid. and dry in an oven at 100-105 DC to constant mass . Carry
Wavelength Use the emission ofbarium at 455.40 nm, out a blank test with 50.0 mL of water jor injections R.
the spectral background being taken at 455 .30 nm. The residue weighs not more than 7.5 mg taking into
Verify the absence of barium in the hydrochloric acid used. account the blank test.
Cadmium Maximum 0.6 ppm. Assay
Atomic absorption spectrometry (2.2.23, Method I) . Carry out the oxygen-flask method (2.5. 10) using 50.0 mg.
Test solution Evaporate 10 mL of solution SI to dryness. Absorb the combustion products in 20 mL of 1 M sodium
Take up the residue using 5 mL of a 1 per cent V/V solution hydroxide. To the solution obtained add 1 mL of dibutyl
of hydrochloric acid R, filter and dilure the filtrate to 10.0 mL phthalate R, 2.5 mL of nitric acid R, 5 mL ofjerric ammonium
with the same acid solurion. suljate solution R2 and 10.0 mL of 0.1 M si/ver nitrate. Titrate
with 0.05 M ammonium thiocyanate until a reddish-yellow
colour is obtained. Carry out a blank test.
cadmium standard solution (0.1 per cent Cd) R, diluting with a
1 per cent VIV solurion of hydrochloric acid R . 1 mL of 0.1 M silver nitrate is equivalent to 6.25 mg of
poly(vinyl chloride).
Source Cadmium hollow-cathode lamp.
In addition, the jollowing tests are carried out on the sterile and
Wavelength 228.8 nm.
empty containers.
Atomisation device Air-acetylene flameo
Solution S3 If the container to be examined contains an
Verify the absence of cadmium in the hydrochloric acid used. anticoagulant solurion, empty the container and wash the
Calcium Maximum 0.07 per cent. inside with 250 mL of water jor injections R at 20 ± 1 oC
Inductively coupled plasma-atomic emission spectrometry and discard the washings before the preparation of solurion
(2.2.57) . S3. Introduce into the container a volume of water jor
injections R corresponding to the volume of solution. Close
V -A548 Appendix XX A 2014
the container and heat in an autoclave so that the without shaking. Remove the container from the water-bath,
temperature of the liquid is maintained at 110 oC for invert it gently 10 times and transfer the contents to a glass
30 mino After cooling, fin the container with water for flask. Irnmediately measure the absorbance at the absorption
injections R to its nominal volume and homogenise. maximum at 272 nm, using the extraction solvent as
Reference solution Heat water for injections R in a compensation liquido
borosilicate-glass flask in an autoclave at 110 cC for 30 mino Determine the concentration of plastic additive O1 in
Reducing substances Immediately after preparation of milligrams per 100 mL of extract from the calibration curve.
solution S3, transfer to a borosilicate-glass flask a volume The concentration does not exceed:
corresponding to 8 per cent of the nominal volume of the - 10 mg per 100 mL for containers of nominal volume
container. At the same time, prepare a blank using an equal greater than 300 mL but not greater than 500 mL;
volume of the freshly prepared reference solution in another - 13 mg per 100 mL for containers of nominal volume
borosilicate-glass flask. To each solution add 20.0 mL of greater than 150 mL but not greater than 300 mL;
0.002 M potassium permanganate and 1 mL of dilute sulfuric
- 14 mg per 100 mL for containers of nominal volume up
acid R. AlIow to stand protected from light for 15 mino
to 150 mL.
To each solution add 0.1 g of potassium iodide R . AlIow to
stand protected from light for 5 min and titrate immediately W'here containers contain an anticoagulant solution, this solution
with O. 01 M sodium thiosulfate, using 0.25 mL of starch complies with the monograph on Anticoagulant and preservative
solution R as indicator. The difference between the two solutions for human blood (0209) and the following additional
titrations is not more than 2.0 mL. test.
Acidity or aIkalinity To a volume of solution S3 Absorbance (2.2.25) Maximum 0.5, by measuring at the
corresponding to 4 per cent of the nominal capacity of the absorption maximum at 280 nm.
container add 0;1 mL of phenolphthalein solution R. Measure the absorbance of the anticoagulant solution from
The solution remains colourless. Add 0.4 mL of 0.01 M the container between 250 nm and 350 nm, using as the
sodium hydroxide. The solution is pink. Add 0.8 mL of compensation liquid an anticoagulant solution of the same
O. 01 M hydrochloric acid and 0.1 mL of methyl red solurion R . composition that has not been in contact with a plastic
The solution is orange-red or red. material.
Ch10rides (2.4.4) Maximum 0.4 ppm, determined on
2. Materials Based on Plasticised Poly(Vinyl
solution S3. Prepare the standard using a mixture of 1.2 mL
Ch1oride) for Tubing Used in Sets for the
of chloride standard solution (5 ppm Cl) R and 13.8 mL of
Transfusion of Blood and Blood Components
water R .
(Ph. Eur. method 3.1.1.2)
Ammonium (2.4.1, Method A) Maximum 2 ppm.
Dilute 5 mL of solution S3 to 14 mL with water R. Definition
Water extractable substances Evaporate 100 mL of Content Minimum 55 per cent of poly(vinyl chloride).
solution S3 to dryness on a water-bath. Dry in an oven to The plasticiser used is di(2-ethylhexyl) phthalate
constant mass at 100-105 oc. Carry out a blank test using (plastic additive 01).
100 mL of the reference solution. The residue from solution Production
S3 weighs not more than 3 mg, taking into account the
Materials based on plasticised poly(vinyl chloride) are
blank test.
produced by polymerisation methods that guarantee a
Absorbance (2.2.25) Maximum 0.30, determined between residual vinyl chloride content of less than 1 ppm.
wavelengths of 230 nm and 250 nm on solution S3; The manufacturing process is validated to demonstrate that
maximum 0.10, determined between wavelengths of 251 nm the product complies with the fonowing test.
and 360 nm on solution S3. Use the reference solution as
Vinyl ch10ride Head-space gas chromatography (2.2.28).
the compensation liquido
Internal standard solution Using a microsyringe, inject
Extractable plastic additive 01 Use as the extraction
10 ¡.tL of ether R into 20.0 mL of dimethylacetamide R,
solvent, ethanol (96 per cent) R diluted with water R to have a
immersing the tip of the needIe in the solvento Immediately
relative density (2.2.5) of 0.9389 to 0.9395, measured with a
before use, dilute the solution to 1000 times its volume with
densimeter.
dimethylacetamide R.
Stock solution Dissolve 0.100 g of plastic additive 01 CRS
Test solution Place 1.000 g of the material to be
in the extraction solvent and dilute to 100.0 mL with the
examined in a 50 mL vial and add 10.0 mL of the internal
same solvent.
Standard solutions Into.5 separate 100 mL volumetric Shake, avoiding contact between the stopper and the liquido
flasks, introduc\,! respectively 1.0 mL, 2.0 mL, 5.0 mL, Place the vial in a water-bath at 60 ± 1 oC for 2 h.
10.0 mL, and 20.0 mL of stock solution.
Vinyl chloride primary solution Prepare urider a
Measure the absorbances (2.2.25) of the standard solutions ventilated hood. Place 50.0 mL of dimethylacetamide R in a
at the absorption maximum at 272 nm, using the extraction 50 mL vial, stopper the vial, secure the stopper and weigh to
solvent as compensation liquid and plot a curve of the nearest 0.1 mg. FilI a 50 mL polyethylene or
absorbance against the concentration of plastic additive 01. polypropylene syringe with gaseous vinyl chloride R, alIow the
Extraction procedure Using the donor tubing and the gas to remain in contact with the syringe for about 3 min,
needle or adapter, fin the empty container with a volume empty the syringe and filI again with 50 mL of gaseous vinyl
equal to half the nominal volume with the extraction solvent, chloride R. Fit a hypodermic needle to the syringe and reduce
previously heated to 37 oC in a welI-stoppered flask. Expel the volume of gas in the syringe from 50 mL to 25 mL.
the air completely from the container and seal the donor Inject the remaining 25 mL of vinyl chloride slowly into the
tubing. Immerse the filled container in a horizontal position vial shaking gently and avoiding contact between the liquid
in a water-bath maintained at 37 ± 1 oC for 60 ± 1 min and the needle. Weigh the vial again; the increase in mas s is
2014 Appendix XX A V -A549
about 60 mg (1 JlL of the solution thus obtained contains Tests

about 1.2 Jlg of vinyl chloride). Allow to stand for 2 h . Keep Jf necessary, cut the samples of the material to be examined into
the primary solution in a refrigerator. pieces with a maximum dimension on a side of not greater than
Vinyl chloride standard solution Vinyl chloride primary 1 cm.
solution, dimethylacetamide R (1:3 VIV) . Solution SI Place 5.0 g of the material to be examined in
Reference solutions Place 10.0 mL ofthe intemal a combustion fiask. Add 30 mL of sulfuric acid R and heat
standard solution in each of six 50 mL vials. Close the vials until a black, syrupy mass is obtained. Cool and add
and secure the stoppers. Inject 1 JlL, 2 JlL, 3 JlL, 5 JlL and carefully 10 mL of strong hydrogen peroxide solution R. Heat
10 JlL, respectively, of the vinyl chloride standard solution gently. Allow to cool and add 1 mL of strong hydrogen
into 5 of the vials. The 6 solutions thus obtained contain peroxide solution R; repeat by altemating evaporation and
respectively, O Jlg, about 0.3 Jlg, 0.6 Jlg, 0.9 Jlg, 1.5 Jlg and addition of hydrogen peroxide solution until a colourless
3 Jlg of vinyl chloride. Shake, avoiding contact between the liquid is obtained. Reduce the volume to about 10 mL. Cool
stopper and the liquido Place the vials in a water-bath at and dilme to 50.0 mL with water R.
60 ± 1 oC for 2 h . Solution S2 Place 25 g of the material to be examined in
Column: a borosilicate-glass fiask. Add 500 mL of water R and cover
- material: stainless steel; the neck of the fiask with a borosilicate-glass beaker. Heat in
an autoclave at 121 ± 2 oC for 20 mino Allow to cool then
- size: 1 = 3 m, 0 = 3 mm;
decant the solution and make up to a volume of 500 mL.
- stationary phase: silanised diatomaceous earth for gas
chromatography R impregnated with 5 per cent mlm of
Appearance of solution S2 Solution S2 is clear (2.2.1)
and colourless (2.2.2, Method JI) .
dimethylstearylamide R and 5 per cent mlm of macrogol
400R. Plastic additive 01 Thin-Iayer chromatography (2.2.27) .
Carrier gas nitrogen for chromatography R. Test solution To 2.0 g of the material to be examined
add 200 mL of peroxide-free ether R and heat under a refiux
Flow rate 30 mUmin.
condenser for 8 h. Separate the residue and the solution by
Temperature: filtration and evaporate the solution to dryness under
- column: 45 oC; reduced pressure in a water-bath at 30 oC . Dissolve the
- injection port: 100 oC; residue in 10 mL of toluene R.
- detector: 150 oc. Reference solution Dissolve 0.8 g of plastic additive
Detection Flame ionisation. 01 CRS in toluene R and dilute to 10 mL with the same
solvent.
lnjection 1 mL of the head space.
Plate TLC silica gel G plate R .
Limit:
Mobile phase toluene R.
- vinyl chloride: maximum 1 ppm.
Application 0.5 mL of the test solution and 5 JlL of the
The supplier of the material must be able to demonstrate
reference solution, as a band 30 mm by 3 mm.
that the qualitative and quantitative composition of the type
sample is satisfactory for each production batch. Development Over a path of 15 cm.
Drying In airo
Characters
Detection In ultraviolet light at 254 nm.
Almost colourless or pale-yellow material in the form of
powder, beads, granules or, after transformation, tubes with a Limit Locate the zone corresponding to plastic additive
slight odour. 01. Remove the area of silica gel corresponding to this zone
and shake with 40 mL of ether R. Filter without loss and
On combustion it gives off dense, black smoke.
evaporate to dryness. The residue weighs not more than
ldentification 40 mg.
Jf necessary, cut the samples of the material to be examined into Barium Maximum 5 ppm.
pie ces with a maximum dimension o'n a side of not greater than Inductively coupled plasma-atomic emission spectrometry
1 cm.
(2.2.57) .
A. To 0.5 g add 30 mL of tetrahydrofuran R. Heat with Test solution Ignite 1.0 g of the substance to be examined
stirring on a water-bath under a hood for 10 mino in a silica crucible. Take up the residue with 10 mL of
The material dissolves completely. Add methanol R hydrochloric acid R and evaporate to dryness on a water-bath.
dropwise with stirring. A granular precipitate is formed. Take up the residue with 20 mL of 0.1 M hydrochloric acid.
Filter the precipitate and dry at 60 oC. Examine the
Reference solution A solution containing 0.25 ppm of
precipitate by infrared absorption spectrophotometry
barium prepared by dilution of barium standard solution
(2.2.24) . Dissolve 50 mg in 2 mL of tetrahydrofuran R
and pour on a glass slide. Dry in an oven at 80 oC, (50 ppm Ea) R with 0.1 M hydrochloric acid.
remove the film and fix on a suitable mount. Examine Wavelength Use the emission of barium at 455.40 nm,
by infrared absorption spectrophotometry (2.2.24), the spectral background being taken at 455.30 nm.
comparing with the spectrum obtained with poly(vinyl Verify the absence of barium in the hydrochloric acid used.
chloride) CRS. Cadmium Maximum 0.6 ppm.
B. Infrared absorption spectrophotometry (2.2.24). Atomic absorption spectrometry (2.2.23, Method J).
Examine the residue obtained in the test plastic additive Test solution Evaporate 10.0 mL of solution SI to
01. dryness. Take up the residue using 5 mL of a 1 per cent VIV
Comparison Plastic additive 01 CRS. solution of hydrochloric acid R, filter and dilute the filtrate to
10.0 mL with the same acid.
V-A550 Appendix XX A 2014
Reference solutions Prepare the reference solutions using potassium permanganate. Boil for 3 min and cool immediately.
eadmium standard solution (0.1 per eent Cd) R, diluting with a Add 1 g of potassium iodide R and titrate with 0.01 M sodium
1 per cent VIV solution of hydroehlorie acid R . thiosulfate using 0.25 mL of stareh solution R as indicator.
Source Cadmium hollow-cathode lampo Carry out a blank test using 20 mL of water for infeetions R.
Wavelength 228.8 nm. The difference between the titration volumes is not greater
than 2.0 mL.
Atomisation device Air-acetylene flameo
Water extractable substances Evaporate 50.0 mL of
Verify the absence of cadmium in the hydrochloric acid used. solution S3 to dryness on a water-bath and dry to constant
Tin Maximum 20 ppm. mass in an oven at 100-105 oC . Carry out a blank test using
Inductively coupled plasma-atomic emission spectrometry 50.0 mL of water for infections R. The residue obtained with
(2.2.57). solution S3 is not greater than 1.5 mg, taking account of the
Test solution Dilute solution SIlO times with water R blank test.
irnmediately before use.
3. Materials Based on Non-plasticised Poly(Vinyl
Reference solution Introduce 2 mL of tin standard solution
CWoride) for Containers for Non-injectable,
(5 ppm Sn) R into a 50 mL flask containing 5 mL of a
Aqueous Solutions
20 per cent V/V solution of sulfurie acid R and dilute to
50 mL with water R immediately before use. (Ph. Eur. method 3.1.10)
Wavelength Use the emission of tin at 189.99 nm, the Definition
spectral background being taken at 190.10 nm. Materials based on non-plasticised poly(vinyl chloride) that
Verify the absence of tin in the sulfuric acid used. comply with the following specifications are suitable for the
Heavy metals (2.4.8) Maximum 50 ppm. manufacture of containers for non-injectable aqueous
solutions. They may also be used for solid forms for oral
To 10 mL of solution SI add 0.5 mL of phenolphthalein
administration and in sorne cases, subject to special studies
solution R and then strong sodium hydroxide solution R until a
on the compatibility of the container with its contents, these
pale pink colour isobtained. Dilute to 25 mL with water R.
materials may be suitable for the preparation of containers
12 mL of the solution complies with test A. Prepare the
for suppositories. They consist of 1 or more poly(vinyl
reference solution using lead standard solution (2 ppm Pb) R.
chloride/vinyl acetate) or of a mixture of poly(vinyl chloride)
Assay and poly(vinyl acetate) or of poly(vinyl chloride).
To 0.500 g add 30 mL of tetrahydrofuran R and heat with The chlorine content expressed as poly(vinyl chloride) is not
stirring on a water-bath under a hood for 10 mino less than 80 per cent.
The material dissolves completely. Add 60 mL of methanol R They may contain not more than 15 per cent of copolymers
dropwise with stirring. A granular precipitate of poly(vinyl based on acrylic and/or methacrylic acids and/or their esters,
chloride) is formed. Allow to stand for a few minutes. and/or on styrene and/or butadiene.
Continue addition of methanol R until no further
precipitation is observed. Transfer to a sintered-glass filter Production
(40) (2.1.2), using three small quantities of methanol R to aid Materials based on non-plasticised poly(vinyl chloride) are
transfer and to wash the precipitate. Dry the filter and the produced by polymerisation methods that guarantee a
precipitate to constant mas s at 60 oC and weigh. residual vinyl chloride content of less than 1 ppm.
In addition, cany out the following tests on sterilised sets. The manufacturing process is validated to demonstrate that
the product complies with the following test.
Solution S3 Make a closed circulation system from 3 sets
and a 300 mL borosilicate-glass vessel. Fit to the vessel a Vinyl chloride Head-space gas chromatography (2.2.28).
suitable thermostat device that maintains the temperature of Internal standard solution Using a microsyringe, inject
the liquid in the vessel at 37 ± 1 oC. Circulate 250 mL of 10 ¡.tL of ether R into 20.0 mL of dimethylaeetamide R,
water for infeetions R through the system in the direction used irnmersing the tip of the needle in the solventoImmediately
for transfusion for 2 h at arate of 1 L!h (for example using a before use, dilute the solution to 1000 times its volume with
peristaltic pump applied to as short a pie ce of suitable dimethylaeetamide R.
silicone elastomer tubing as possible) . Collect the whole of Test solution Place 1.000 g of the material to be
the solutión and allow to cool. examined in a 50 mL vial and add 10.0 mL of the internal
Appearance ofsolution Solution S3 is clear (2.2.1) and standard solution. Close the vial and secure the stopper.
colourless (2.2.2, Method 11). Shake, avoiding contact between the stopper and the liquido
Acidity or alkalinity To 25 mL of solution S3 add Place the vial in a water-bath at 60 ± 1 oC for 2 h.
0.15 mL of BRP indicator solution R . Not more than 0.5 mL Vinyl chloride primary solution Prepare under a
of 0.01 M sodium hydroxide is required to change the colour ventilated hood. Place 50.0 mL of dimethylaceramide R in a
of the indicator to blue. To 25mL of solution S3 add 50 mL vial, stopper the vial, secure the stopper and weigh to
0.2 mL of methyl orange solution R. Not more than 0.5 mL of the nearest 0.1 mg. Fill a 50 mL polyethylene or
O. 01 M hydroehlorie aeid is required to initiate the colour polypropylene syringe with gaseous vinyl ehloride R, allow the
change of the indicator from yellow to orange. gas to remain in contact with the syringe for about 3 min,
Absorbance (2.2.25) Maximum 0.30, determined between empty the syringe and fill again with 50 mL of gaseous vinyl
wavelengths of 230 nm and 250 nm on solution S3; ehloride R. Fit a hypodermic needle to the syringe and reduce
maximum 0.15, determined between wavelengths of 251 nm the volume of gas in the syringe from 50 mL to 25 mL.
and 360 nm on solution S3. Inject these 25 mL of vinyl chloride slowly into the vial,
shaking gently and avoiding contact between the liquid and
the needle. Weigh the vial again; the increase in mas s is
preparation of solution S3. To 20.0 mL of solution S3 add
about 60 mg (1 ¡.tL of the solution thus obtained contains
1 mL of dilute sulfurie aeid R and 20.0 mL of 0.002 M
about 1.2 ¡.tg of vinyl chloride). Allow to stand for 2 h. Keep - 1-phenyleicosane-1,3-dione (benzoylstearoylmethane) or
the primary solution in a refrigerator. 2-( 4-dodecylphenyl)indole or didodecyl
Vinyl chloride standard solution Vinyl chloride primary 1,4-dihydropyridine-2, 6-dimeth yl-3 ,5-dicarboxylate:
solution, dimethylacetamide R (1:3 V/V). maximum 1 per cent or 1 per cent of a mixture of two of
these.
Reference solutions Place 10.0 mL of the intemal
standard solution in each of six 50 mL vials. Close the vials They may contain a colorant or pigment and may be
and secure the stoppers. Inject 1 ¡.tL, 2 ¡.tL, 3 ~lL, 5 ¡.tL and opacified by titanium dioxide.
10 ¡.tL, respectively, of the vinyl chloride standard solution The supplier of the material must be able to demonstrate
into 5 of the vials. The 6 solutions thus obtained contain that the qualitative and quantitative composition of the type
respectively, O ¡.tg, about 0.3 ¡.tg, 0.6 ¡.tg, 0.9 ¡.tg, 1.5 ¡.tg and sample is satisfactory for each production batch.
3 ¡.tg of vinyl chloride. Shake, avoiding contact between the Characters
stopper and the liquid. Place the vials in a water-bath at
60 ± 1 oC for 2 h. Appearance Powder, beads, granules, sheets of varying
thicknesses or samples taken from finished objects.
Column:
Solubility Insoluble in water, soluble in tetrahydrofuran,
- material: stainless steel; slightly soluble in methylene chloride, insoluble in anhydrous
- size: 1 = 3 m, 0 = 3 mm; ethano!.
- stationary phase: silanised diatomaceous earth for gas They bum with an orange-yellow fiame edged with green,
chromatography R impregnated with 5 per cent m/m of giving off thick black smoke.
dimethylstearylamide R and 5 per cent m/m of macrogol
Identification
400 R.
Infrared absorption spectrophotometry (2.2.24).
Carner gas nitrogen for chromatography R.
Preparation Dissolve the residue A (see Tests: solution
Flow rate 30 mUmin.
S2) in 5 mL of tetrahydrofuran R. Apply a few drops of the
Temperature: solution to a sodium chloride plate and evaporate to dryness
- column: 45 oC; in an oven at 100-105 oc.
- injection port: 100 oC; Absorption maxima At 2975 cm- I , 2910 cm- I ,
- detector: 150 cc. 2865 cm- \ 1430 cm- I , 1330 cm- \ 1255 cm- I , 690 cm- I
Detection Flame ionisation. and 615 cm- l.
Injection 1 mL of the head space. The spectrum obtained is identical to that of the material
selected for the type sample.
Limit:
- vinyl chloride: maximum 1 ppm. TESTS
Jf necessary, cut the samples of the material to be examined into
ADDITIVES
pieces with a maximwn dimension on a side of not greater than
In order to obtain the required mechanical and stability 1 cm.
characteristics, materials based on non-plasticised poly(vinyl
Solution S1 Place 25 g in a borosilicate-glass fiask.
chloride) may contain:
Add 500 mL of water R and cover the neck of the fiask with
- epoxidised soya oil of which the oxiran oxygen content is aluminium foil or a borosilicate-glass beaker. Heat in an
6 per cent to 8 per cent and the iodine value is not greater autoclave for 121 ± 2 oC for 20 mino Allow to cool and
than 6: maximum 8 per cent; allow the solids to settle.
- calcium salt or zinc salts of aliphatic fatty acids with more Solution S2 Dissolve 5.0 g in 80 mL of tetrahydrofuran R
than 7 carbon atoms: maximum 1.5 per cent or maximum and dilute to 100 mL with the same solvento Filter if
1.5 per cent of their mixture; necessary (the solution may remain opalescent). To 20 mL
- liquid paraffin: maximum 1.5 per cent; of the solution add, dropwise and with gentle shaking,
- waxes: maximum 1.5 per cent; 70 mL of ethanol (96 per cent) R. ' Cool in ice for 1 h. Filter
- hydrogenated oils or esters of aliphatic fatty acids: or centrifuge (residue A). Wash the residue A with ethanol
maximum 2 per cent; (96 per cent) R , add the washings to the filtrate or the
centrifugation liquid and, dilute to 100 mL with ethanol
- macrogol esters: maximum 1.5 per cent;
(96 per cent) R.
- sorbitol: maximum 1.5 per cent;
Solution S3 Place 5 g in a borosilicate-glass fiask with a
- 2,4-dinonylphenyl phosphite, or di(4-nonylphenyl) ground-glass neck. Add 100 mL of 0.1 M hydrochloric acid
phosphite 'or tris(nonylphenyl) phosphite: maximum and boil under a refiux condenser for 1 h. Allow to cool and
1 per cent. allow the solids to settle.
They may contain one of the following groups of stabilisers: Appearance of solution S1 Solution SI is not more
- tin as di(isooctyl) opalescent than reference suspension 11 (2.2.1) and is
2,2'- [( dioctylstannylene) bis(thio))diacetate containing colourless (2.2.2, Method Il) .
about 27 per cent of tri(isooctyl) 2,2 ',2"- Absorbance of solution S 1 (2.2.25) Evaporate 100 mL of
[(monooctylstannylidyne )tris (thio») triacetate: maximum solution SIto dryness. Dissolve the residue in 5 mL of
0.25 per cent; hexane R . Filter if necessary through a filter previously rinsed
- tin as a mixture containing not more than 76 per cent of with hexane R. At wavelengths from 250 nm to 310 nm, the
di(isooctyl) 2,2'-[(dimethylstannylene)bis(thio»)diacetate absorbance of the filtra te is not greater than 0.25.
and not more than 85 per cent oftri(isooctyl) 2,2',2"- Absorbance of solution S2 (2.2.25) Maximum 0.2 for
[(monomethylstannylidyne)tris(thio») triacetate; (isooctyl is tin-stabilised materials or 0.4 for other materials determined
e.g. 2-ethylhexyl): maximum 0.25 per cent; between wavelengths of 250 nm and 330 nm on solution S2.
V-A552 Appendix xx A 2014
Extractable barium Maximum 2 ppm. Reference solution A solution containing 0.50 ppm of
Inductively coupled plasma-atomic emission spectrometry zinc prepared by dilution of zinc standard solution (5 mg/mL
(2.2.57). Zn) R with 0.01 M hydrochloric acid.
Test solution Solution S3. Verify the absence of zinc in the hydrochloric acid used.
Reference solution A solution containing 0.1 ppm of Examined at 214 .0 nm, the absorbance of the test solution is
barium prepared by dilution of barium standard solution not greater than that of the reference solution.
(50 ppm Ea) R with 0.1 M hydrochloric acid. Sulfated ash (2.4.14) Maximum 1.0 per cent, determined
Wavelength Use the emission of barium at 455.40 nm, on 1.0 g; maximum 4.0 per cent when the materials are
the spectral background being taken at 455 .30 nm. opacified using titanium dioxide.
Verify the absence of barium in the hydrochloric acid used. Assay
Examined at 455.40 nm, the emission of the test solution is Carry out the oxygen-flask method (2.5.10) using 50.0 mg of
not greater than that of the reference solution. the material to be examined. Absorb the combustion
Extractable cadnúum Maximum 0.6 ppm. products in 20 mL of 1 M sodium hydroxide. To the solution
obtained add 1 mL of dibutyl phthalate R, 2.5 mL of nitric
Atomic absorption spectrometry (2.2.23, Method ¡).
acid R, 5 mL ofjerric ammonium sulfate solution R2 and
Test solution Solution S3 . 10.0 mL of 0.1 M si/ver nitrate. Titrate with 0.05 M
Reference solution A solution containing 0.03 ppm of ammonium thiocyanate until a reddish-yellow colour is
cadmium prepared by diluting cadmium standard solution obtained. Carry out a blank titration.
(O. 1 per cent Cd) R with 0. 1 M hydrochloric acid. 1 mL of 0. 1 M silver nitrate is equivalent to 6.25 mg of
Verify the absence of cadmium in the hydrochloric acid used. poly(vinyl chloride).
Examined at 228.8 nm, the absorbance of the test solution is
not greater than that of the reference solution. 4. Materials Based on Non-plasticised Poly(Vinyl
Tin-stabilised materials Maximum 0.25 per cent of Sn. Chloride) for Dry Dosage Forms for Oral
Administration
Tin stock solution Dilute 81 mg of plastic additive
23 CRS to 100.0 mL with tetrahydrojuran R . (Ph. Eur. method 3.1. 11)
Tin standard solution Dilute 20 mL of the tin stock Definition
solution to 100:0 mL with ethanol (96 per cent) R . Materials based on non-plasticised poly(vinyl chloride) for
To 0.10 mL ofsolution S2 in a test tube add 0.05 mL of containers for dry dosage forms for oral administration are
1 M hydrochloric acid, 0.5 mL of potassium iodide solution R suitable for the manufacture of sheets or containers, and
and 5 mL of ethanol (96 per cent) R. Mix thoroughly and wait consist of 1 or more poly(vinyl chloride/vinyl acetate) or of a
for 5 mino Add 9 mL of water R and 0.1 mL of a 5 gIL mixture of poly(vinyl chloride) and poly(vinyl aceta te) or of
solution of sodium sulfite R and mix thoroughly. Add 1.5 mL poly(vinyl chloride).
of dithizone solution R freshly diluted 100-fold with methylene The chlorine content expressed as poly(vinyl chloride) is not
chloride R, shake for 15 s and allow to stand for 2 mino At the les s than 80 per cent.
same time prepare a reference solution in the same manner They may contain not more than 15 per cent of copolymers
using 0.1 mL of the tin standard solution. based on acrylic and/or methacrylic acids and/or their esters,
Any violet colour in the lower layer obtained with solution S2 and/or on styrene and/or butadiene.
is not more intense than that obtained with the reference
Production
solution. The greenish-blue colour of dithizone solution turns
pink in the presence of tino Materials based on non-plasticised poly(vinyl chloride) are
produced by polymerisation methods that guarantee a
Non-tin stabilis~d materials Maximum 25 ppm of Sn.
residual vinyl chloride content of less tlfan 1 ppm.
To 5 mL of solution S2 in a test tube add 0.05 mL of 1 M The manufacturing process is validated to demonstrate that
hydrochloric acid and 0.5 mL of potassium iodide solution R. the product complies with the following test for vinyl
Mix thorbughly and wait for 5 min oAdd 9 mL of water R chloride.
and 0.1 mL of a 5 gIL solution of sodium sulfite R and mix
Vinyl chloride Head-space gas chromatography (2.2.28).
thoroughly. If the solution obtained is not colourless, add the
sodium sulfite solution in 0.05 mL fractions . Add 1.5 mL of Internal standard solution Using a microsyringe, inject
dithizone solution R freshly diluted 100-fold with methylene 10 IlL of ether R into 20.0 mL of dimethylacetamide R,
chloride R, shake for 15 s and allow to stand for 2 mino At the immersing the tip of the needle in the solvent. Immediately
same time prepare a reference solution in the same manner before use, dilute the solution to 1000 times its volume with
using 0.05 inL of the tin standard solution (see test aboye). dimethylacetamide R.
Any violet colour in the lower layer obtained with solution S2 Test solution Place 1.000 g of the material to be
is not more intense than that obtained with the reference examined in a 50 mL vial and add 10.0 mL of the internal
solution. standard solution. Close the vial and secure the stopper.
Shake, avoiding contact between the stopper and the liquido
Extractable heavy metals (2.4.8) Maximum 20 ppm.
Place the vial in a water-bath at 60 ± 1 oC for 2 h.
12 mL of solution S3 complies with test A. Prepare the
Vinyl chloride primary solution Prepare under a
reference solution using 10 mL of lead standard solution
ventilated hood. Place 50.0 mL of dimethylacetamide R in a
(1 ppm Pb) R .
Extractable zinc Maximum 100 ppm. the nearest 0.1 mg. Fill a 50 mL polyethylene or
Atomic absorption spectrometry (2.2.23, Method ¡). polypropylene syringe with gaseous vinyl chloride R , allow the
Test solution Solution S3 diluted 10-fold with water R. gas to remain in contact with the syringe for about 3 min,
empty the syringe and fill again with 50 mL of gaseous vinyl
chloride R . Fit a hypodermic needle to the syringe and reduce
the volume of gas in the syringe from 50 mL ro 25 mL. ~ silica: maximum 1 per cent.
Inject the 25 mL of vinyl chloride slowly into the vial, They may contain one of the 3 following groups of stabilisers
shaking gently and avoiding contact between the liquid and (where isooctyl is, for example, 2-ethylhexyl):
the needle. Weigh the vial again; the increase in mass is ~ tin as di(isooctyl)
about 60 mg (1 flL of the solution thus obtained contains 2,2' - [( dioctylstannylene) bis (thio )] diacetate containing
about 1.2 ~lg of vinyl chloride). Allow to stand for 2 h. Keep
about 27 per cent of tri (isooctyl) 2,2 '2 "-
the primary solution in a refrigeraror.
[(monooctylstannylidyne)tris (thio)] triacetate: maximum
Vinyl chloride standard solution vinyl chloride primary 0.25 per cent;
solution, dimethylacetamide R (1:3 V/V).
~ tin as a mixture containing not more than 76 per cent of
Reference solutions Place 10.0 mL of the intemal di(isooctyl) 2,2'-[( dimethylstannylene)bis (thio)] diacetate
standard solution in each of six 50 mL vials. Close the vials and not more than 85 per cent of tri(isooctyl) 2,2',2"-
and secure the sroppers. Inject 1 flL, 2 flL, 3 flL, 5 pL and [(monomethylstannylidyne)tris(thio)]triacetate: maximurn
10 pL, respectively, of the vinyl chloride standard solution 0.25 per cent;
into 5 of the vials. The 6 solutions thus obtained contain ~ 1-phenyleicosane-1,3-dione (benzoylstearoylmethane):
respectively, O flg, about 0.3 flg, 0.6 flg, 0.9 ~lg, 1.5 ¡.¡g and maximum 1 per cent.
3 ¡.¡g of vinyl chloride. Shake, avoiding contact between the
stopper and the Iiquid. Place the vials in a water-bath at They may contain a colorant or pigment and may be
60 ± 1 oC for 2 h. opacified by titanium dioxide.
Column
that the qualitative and quantitative composition of the type
~ material: stainless steel; sample is satisfactory for each production batch.
~ size: 1 = 3 m, 0 = 3 mm;
Characters
~ stationary phase: silanised diatomaceous earth for gas
chromatography R impregnated with 5 per cent rn/m of Appearance Powder, beads, granules, sheets of varying
thicknesses or samples taken from finished objects.
dimethylstearylamide R and 5 per cent rn/m of macrogol
400 R. Solubility Insoluble in water, soluble in tetrahydrofuran,
slightly soluble in methylene chloride, insoluble in anhydrous
Carrier gas nitrogen for chromatography R.
ethano!.
Flow rate 30 mUmin.
They bum with an orange-yellow fiame edged with green,
Tempera tu re giving off thick black smoke.
~ column: 45 oC;
1dentification
~ injection pore: 100 oC;
Infrared absorption spectrophotometry (2.2.24).
~ detector: 150 ce.
Preparation Dissolve residue A (see Tests: solution S2) in
Detection Flame ionisation. 5 mL of tetrahydrofuran R. Apply a few drops of the solution
Injection 1 mL of the head space. to a sodium chloride plate and evaporate to dryness in an
Limit oven at 100-105 ce.
~ vinyl ch/oride: maximurn 1 ppm. Absorption maxima At 2975 cm- l, 2910 cm- l ,
ADDITIVES
2865 cm- l, 1430 cm- l, 1330 cm- l, 1255 cm- l , 690 cm- l
and 615 cm- l.
In order ro obtain the required mechanical and stability
characteristics, materials based on non-plasticised poly(vinyl The spectrum obtained is identical to that of the material
chloride) may contain: selected for the type sample.
~ epoxidised soya oil of which the oxiran oxygen content is Tests
6 per cent to 8 per cent and the iodine value is not greater [f necessary, cut the samples of the materialto be examined into
than 6 for tin-stabilised materials: maximum 2 per cent; pieces with a maximum dimension on a side of n01 greater than
~ epoxidised soya oil of which the oxiran oxygen content is 1 cm.
6 per cent to 8 per cent and the iodine value is not greater Soludon SI Place 25 g in a borosilicate-glass fiask.
than 6 for non-tin-stabilised materials: maximurn Add 500 mL of water R and cover the neck of the fiask with
3 per cent; aluminium foil or a borosilicate glass beaker. Heat in an
~ ca1cium, magnesium or zinc salts of aliphatic fatty acids autoclave for 121 ± 2 oC for 20 mino Allow to cool and
with more than 7 carbon atoms: maximum 1.5 per cent or allow the solids to settle.
maximum 1.5 ·per cent of their mixture; Solution S2 Dissolve 5.0 g in 80 mL of tetrahydrofuran R
~ waxes: malj:imum 4 per cent; and dilute to 100 mL with the same solvent. Filter if
~ Iiquid paraffin: maximum 1.5 per cent; necessary (the solution may remain opalescent). To 20 mL
of the solution add, dropwise and with gentle shaking,
~ hydrogenated oils or esters of aliphatic fatty acids:
70 mL of ethanol (96 per cent) R. Cool in ice for 1 h. Filter
maximum 2 per cent;
or centrifuge (residue A). Wash residue A with ethanol
~ the percentage sum of the 3 lubricants aboye: maximum
(96 per cene) R, add the washings to the filtrate or the
4 per centj centrifugation liquid and dilute to 100 mL with ethanol
~ macrogol esters: maximum 1.5 per cent; (96 per cene) R.
~ sorbitol: maximum 1.5 per cent; Solution S3 Place 5 g in a borosilicate-glass fiask with a
~ 2,4-dinonylphenyl phosphite, or di(4-nonylphenyl) ground-glass neck. Add 100 mL of 0.1 M hydrochloric acid
phosphite or tris(nonylphenyl) phosphite: maximum and boil under a refiux condenser for 1 h . Allow to cool and
1 per cent; allow the solids to settle.
~ ca1cium carbonate: maximum 1 per cent;
Appearance of solution S1 Solution SI is not more Assay

opalescent than reference suspension II (2.2.1) and is Carry out the oxygen-flask method (2.5. JO) using 50.0 mg of
colourless (2.2.2, Method JI) . the material to be examined. Absorb the combustion
Absorbance ofsolution S1 (2.2.25) Evaporate 100 mL of products in 20 mL of 1 M sodium hydroxide. To the solution
solution SIto dryness. Dissolve the residue in 5 mL of obtained add 2.5 mL of nitric acid R, 10.0 mL of 0. 1 M silver
hexane R. Filter if necessary through a filter previously rinsed nitrate, 5 mL of jerric ammonium sulfate solution R2 and 1 mL
with hexane R. At wavelengths from 250 nm to 310 nm, the of dibutyl phthalate R. Titrate with 0.05 M ammonium
absorbance of the filtrate is not greater than 0.3. thiocyanate until a reddish-yellow colour is obtained. Carry
Absorbance of solution S2 (2.2.25) Maximum 1.0, out a blank titration.
determined between wavelengths of 250 nm and 330 nm on 1 mL of 0. 1 M silver nitrate is equivalent to 6.25 mg of
solution S2 for material that does not contain poly(vinyl chloride).
l-phenyleicosane-l,3-dione; maximum 0.4, determined
between wavelengths of 250 nm and 330 nm on a 10-fold 5. Materials Based on Plasticised Poly(Vinyl
dilution of solution S2 in ethanol (96 per cent) R for material Ch1oride) for Containers for Aqueous Solutions for
that contains l-phenyleicosane-l,3-dione. Intravenous Infusion
Tin-stabilised materials Maximum 0.25 per cent of Sn. (Ph. Eur. method 3.1.14)
Tin stock solution Dilute 81 mg of plastic additive Definition
23 CRS to 100.0 mL with tetrahydrofuran R. Materials based on plasticised poly(vinyl chloride) contain
Tin standard solution Dilute 20 mL of the tin stock not less than 55 per cent of poly(vinyl chloride) and contain
solution to 100.0 mL with ethanol (96 per cent) R. various additives, in addition to the high-molecular-mass
To 0.10 mL ofsolution S2 in a test tube add 0.05 mL of polymer obtained by polymerisation of vinyl chloride.
1 M hydrochloric acid, 0.5 mL of potassium iodide solution R Materials based on plasticised poly(vinyl chloride) for
and 5 mL of ethanol (96 per cent) R. Mix thoroughly and wait containers for aqueous solutions for intravenous infusion are
for 5 mino Add 9 mL of water R and 0.1 mL of a 5 gIL defined by the nature and the proportions of the substances
solution of sodium sulfite R and mix thoroughly. Add 1.5 mL used in their manufacture.
of dithizone solution R freshly diluted 100-fold with methylene Production
chloride R, shake for 15 s and allow to stand for 2 mino
Materials based on plasticised poly(vinyl chloride) are
At the same time prepare a reference solution in the same
produced by polymerisation methods which guarantee a
manner using 0.1 mL of the tin standard solution.
residual vinyl chloride content of les s than 1 ppm.
Any violet colour in the lower layer obtained with solution S2 The production method used is validated in order to
is not more intense than that obtained with the reference demonstrate that the product complies with the following
solution. The greenish-blue colour of dithizone solution tums test.
pink in the presence of tino
VinyI chloride Head space gas chromatography (2.2.28).
Non-tin-stabilised materials Maximum 25 ppm of Sn.
lnternal standard solution Using a microsyringe, inject
To 5 mL of solution S2 in a test tube add 0.05 mL of 1 M 10 ¡.tL of ether R into 20.0 mL of dimethylacetamide R,
hydrochloric acid and 0.5 mLof potassium iodide solution R. immersing the tip of the needle in the solvent. Immediately
Mix thoroughly and wait for 5 mino Add 9 mL of water R before use, dilute the solution to 1000 times its volume with
and 0.1 mL of a 5 gIL solution of sodium sulfite R and mix dimethylacetamide R.
thoroughly. If the solution obtained is not colourless, add tbe
Test solution Place 1.000 g of the material to be
sodium sulfite solution in 0.05 mL fractions. Add 1.5 mL of
examined in a 50 mL vial and add 10.0 mL of the intemal
dithizone solution R freshly diluted 100-fold with methylene
chloride R, shake for 15 s and allow to stand for 2 mino At the
Shake, avoiding contact between the stopper and the liquid.
same' time. prepare a reference solution in the same manner
Place the vial in a water-bath at 60 ± 1 oC for 2 h .
using 0.05 mL of the tin standard solution (see test aboye).
Vinyl chloride primary solution Prepare under a
Any violet colour in the lower layer obtained with solution S2
ventilated hood. Place 50.0 mL of dúnethylacetamide R in a
isnot more intense than thar- obtained with the reference
solution.
the nearest 0.1 mg. Fill a 50 mL polyethylene or
Extractable heavy metals (2.4.8) Maximum 20 ppm. polypropylene syringe with gaseous vinyl chloride R, allow the
12 mL of solution S3 complies with test A. Prepare the gas to remain in contact with the syringe for about 3 min,
reference solution using 10 mL of lead standard solution empty the syringe and fill again with 50 mL of gaseous vinyl
(1 ppm Pb) R. chloride R. Fit a hypodermic needle to the syringe and reduce
Extractable zinc Maximum 100 ppm. the volume of gas in the syringe from 50 mL to 25 mL.
Atomic absorption spectrometry (2.2.23, Method I) . Inject tbe remaining 25 mL of vinyl chloride slowly into the
vial shaking gently and avoiding contact between the liquid
Test solution Solution S3 diluted lO-fold with water R .
and the needle. Weigh the vial again; the increase in mass is
Reference solution A solution containing 0.50 ppm of about 60 mg (1 ¡.tL of the solution thus obtained contains
zinc prepared by dilution of zinc standard solution (5 mg/mL about 1.2 ¡.tg of vinyl chloride) . Allow to stand for 2 h. Keep
Zn) R with O. 01 M hydrochloric acid. the primary solution in a refrigerator.
Verify the absence of zinc in the hydrochloric acid used. Vinyl chloride standard solution Vinyl chloride primary
Examined at 214.0 nm, the absorbance ofthe test solution is solution, dimethylacetamide R (l :3 V/V).
not greater than that of the reference solution. Reference solutions Place 10.0 mL of the intemal
Sulfated ash (2.4.14) Maximum 1.0 per cent, determined standard solution in each of six 50 mL vials. Close tbe vials
on 1.0 g; maximum 4.0 per cent when the materials are and secure tbe stoppers. Inject 1 ¡.tL, 2 ¡.tL, 3 ¡.tL, 5 ¡.tL and
opacified using titanium dioxide. 10 ¡.tL, respectively, of the vinyl chloride standard solution
into 5 of the vials. The 6 solutions thus obrained contain, Identification

respectively, O ~tg, about 0.3 ¡.tg, 0.6 ¡.tg, 0.9 ~tg, 1.5 ¡.tg and Jf necessary, before use, cut the samples of the material to be
3 ¡.tg of vinyl chloride. Shake, avoiding contact between the examined into pieces of maximum dimension on a side of not
stopper and the liquido Place the vials in a water-bath at greater than 1 cm.
60 ± 1 oC for 2 h. To 2.0 g of the material 10 be examined add 200 mL of
Column: peroxide-free ether R and heat under a refiux condenser for
- material: stainless steel; 8 h. Separate the residue E and the solution A by filtration.
- size: l = 3 m, 0 = 3 mm; Evaporate solution A 10 dryness under reduced pressure in a
- stationary phase: silanised diatomaceous earth for gas water-bath at 30 oc. Dissolve the residue in 10 mL of
chromatography R impregnated with 5 per cent m /m of toluene R (solution Al) . Dissolve the residue E in 60 mL of
dimethylstearylamide R and 5 per cent m /m of macrogol ethylene chloride R, heating on a water-bath under a refiux
400R. condenser. Filter. Add the obtained solution dropwise and
with vigorous shaking to 600 mL of heptane R heated almost
Carner gas nitrogen for chromatography R.
10 boiling. Separate by hot filtration the coagulum El and
Flow rate 30 mUmin. the organic solution. AlIow the latter to cool; separate the
Temperature: precipitate E2 that forms and filter through a tared
- column: 45 oC; sintered-glass filter (40) (2. 1.2) .
- injection port: 100 DC; A. Infrared absorPtion spectrophotometry (2.2.24) .
- detector. 150 oC . Preparation Dissolve the coagulum El in 30 mL of
Detection Flame ionisation. tetrahydrofuran R and add, in smalI volumes with shaking,
40 mL of anhydrous ethanol R. Separate the precipitate E3 by
Injection 1 mL of the head-space.
filtration and dry in vacuo at a temperature not exceeding
Limit: 50 oC over diphosphorus pentoxide R. Dissolve a few
- vinyl chloride: maximum 1 ppm. milligrams of precipitate E3 in 1 mL of tetrahydrofuran R,
ADDITIVES place a few drops of the solution obtained on a sodium
A certain number of additives is added to the polymers 10 chloride plate and evaporate 10 dryness in an oven at
optimise their chemical, physical and mechanical properties 100-105 oC.
in order to adapt them for the intended use. AlI these Comparison poly(vinyl chloride) CRS.
additives are chosen from the following list which specifies E. Infrared absorPtion spectrophotometry (2.2.24).
for each product the maximum alIowable content: Examine the residue C obtained in the test for plastic
- di(2-ethylhexyl)phthalate (plastic additive 01): maximum additives 01, 04 and 05.
40 per cent; Comparison plastic additive 01 CRS.
- zinc octanoate (zinc 2-ethylhexanoate) (plastic additive
02) : maximum 1 per cent;
Tests
Jf necessary, before use, cut the samples of the material to be
- calcium stearate or zinc stearate: maximum 1 per cent or
examined into pie ces of maximum dimension on a side of not
1 per cent of a mixture of the two;
greater than 1 cm.
- N,N'-diacylethylenediamines (plastic additive 03):
Solution SI Place 5.0 g in a combustion fiask.
maximum 1 per cent;
Add 30 mL of sulfuric acid R and heat until a black, syrupy
- maximum 10 per cent of one of the following epoxidised mas s is obtained. Cool and add carefully 10 mL of strong
oils or 10 per cel).t of a mixture of the two: hydrogen peroxide solution R. Heat gently. Allow to cool and
- epoxidised soya oil (plastic additive 04) of which the add 1 mL of strong hydrogen peroxide solution R; repeat by
oxiran oxygen content is 6 per cent 10 8 per cent and altemating evaporation and addition of strong hydrogen
the iodine value is not greater than 6; peroxide solution until a colourIess liquid is obtained.
- epoxidised linseed oil (plastic additive 05) of which the Reduce the volume to about 10 mL. Cool and dilute to
oxiran oxygen content is not greater than 10 per cent 50.0 mL with water R.
and the iodine value is not greater than 7. Solution S2 Place 25 g in a borosilicate-glass fiask.
When colouring materials are added, ultramarine blue is Add 500 mL of water for injections R and cover the neck of
used. Other inorganic pigments may be added, provided the the fiask with aluminium foil or a borosilicate-glass beaker.
safety of the material is demonstrated 10 the satisfaction of Heat in an autoclave at 121 ± 2 oC for 20 mino Allow to
the competent authority. Very low amounts of antioxidants cool and decant the solution.
added 10 the vinyl chloride monomer used may be detected Appearance of solution S2 Solution S2 is clear (2.2.1)
in the polyrner. and colourless (2.2.2, Method 11).
The supplier of the material must be able 10 demonstrate Acidity or aIkalinity To 100 mL of solution S2, add
that the qualitative and quantitative composition of the type 0.15 mL of BRP indicator so/ution R . Not more than 1.5 mL
sample is satisfac10ry for each production batch. of 0.01 M sodium hydroxide is required to change the colour
Characters of the indicator 10 blue. To 100 mL of solution S2 add
0.2 mL of methyl orange solution R. Not more than 1.0 mL of
ColourIess or pale yellow material in the form of powder,
0.01 M hydrochloric acid is required to initiate the colour
beads, granules or, after transformation, translucent sheets of
change of the indicator from yellow to orange.
varying thicknesses, with a slight odour. On combustion it
gives off dense, black smoke. Absorbance (2.2.25) Evaporate 100.0 mL of solution
S2 to dryness. Dissolve the residue in 5.0 mL of hexane R.
From 250 nm 10 310 nm the absorbance is not greater
than 0.25.
Reducing substances Carry out the test within 4 h of Inductively coupled plasma-atomic emission spectrometry
preparatiol1 of solutiol'l S2. To 20.0 mL of solution S2 add (2.2.57) .
1 mL of dilme SUlfUlic acid R and 20. O mL of 0.002 M Test solution Ignite 1.0 g of the substance to be examined
potassium pennanganate. Boil under a reflux condenser for in a silica crucible. Take up the residue with 10 mL of
3 min and cool immediately. Add 1 g of pocassium iodide R hydrochloric acid R and evaporate ro dryness on a water-bath.
and ti trate immediately with 0.01 M sodium thiosulfare, using Take up the residue with 20 mL of 0.1 M hydrochloric acid.
0.25 mL of starch solution R as indicator. Carry out a blank Reference solution A solution containing 0.25 ppm of
titration using 20 mL of water for injectiol1s R. The difference barium prepared by dilution of barium standard solution
between rhe titration volumes is not more rhan 2.0 mL. (5 0 ppm Ea) R with 0.1 M hydrochlO1ic acid.
Primary aromatic amines Maximum 20 ppm. Wavelength Use the emission of barium at 455.40 nm,
To 2.5 mL of solution Al obtained during the identification, the spectral background being taken at 455.30 nm.
add 6 mL of water R and 4 mL of 0.1 M hydrochlolic acid. Verify the absence of barium in the hydrochloric acid used.
Shake vigorously and discard the upper layer. To the lower
Cadmium Maximum 0.6 ppm.
layer add 0.4 mL of a freshly prepared 10 gIL solution of
sodium nitrite R. Mix and allow ro stand for 1 mino Aromic absorption spectrometry (2.2.23, Method ¡).
Add 0.8 mL of a 25 gIL solution of ammoniUln sulfamate R, Test solution Evaporate 10 mL of solution S 1 ro dryness.
allow ro stand for 1 min and add 2 mL of a 5 gIL solution of Take up the residue using 5 mL of a 1 per cent V/V solution
naphthylethylenediamine dihydrochloride R . After 30 min, any of hydrochloric acid R, filter and dilute the filtrate ro 10.0 mL
colour in the solution is not more intense than that in a with the same acid.
standard prepared at the same time in the same manner Reference solutions Prepare the reference solutions using
using a mixture of 1 mL of a 0.01 gIL solution of cadmium standard solution (0.1 per cent Cd) R, diluting with a
naphthylamine R in O. 1 M hydrochloric acid, 5 mL of water R 1 per cent VIV solution of hydrochloric acid R.
and 4 mL of 0.1 M hydrochloric acid instead of the aqueous
Source Cadmium hollow-cathode lampo
layer.
P1astic additives 01, 04 and 05 Thin-layer
chromatography (2.2.27). Atomisation device Air-acetylene flame o
Reference solutions Prepare 0.1 mg/mL solutions of Verify the absence of cadmium in the hydrochloric acid used.
plastic addüive 01 CRS, plastic additive 04 CRS and plastic Calcium Maximum 0.07 per cent.
additive 05 CRS, respectively, in lOluene R. Inductively coupled plasma-atomic emission spectrometry
Plate TLC silica gel GF254 plate R. (2.2.57).
Mobile phase lOluene R. Test solution Use the test solution prepared for the
Application O.? mL of solution Al obtained during the determination of barium.
identification as a band 30 mm by 3 mm and 5 ¡.¡L of each Reference solution A solution containing 50.0 ppm of
reference solution. calcium prepared by dilution of calcium standard solution
Development Over a path of 15 cm. (400 ppm Ca) R with 0.1 M hydrochloric acid.
Drying In airo Wavelength Use the emission of calcium at 315.89 nm,
the spectral background being taken at 315.60 nm.
Detection A · Examine in ultraviolet light at 254 nm.
Verify the absence of calcium in the hydrochloric acid used.
Locate the zone corresponding to plastic additive 01 (R p
about 0.4). Remove the area of silica gel corresponding ro Tin Maximum 20 ppm.
this zone and shake with 40 mL of ether R for 1 mino Filter, Inductively coupled plasma-atomic emission spectrometry
rinse with 2 quantities, each of 10 mL of ether R, add the (2.2.57).
rinsings ro the filtrate and evaporate ro dryness. The residue Test solution Dilute solution SIlO times with water R
C weighs not more than 40 mg. immediately before use.
Detection B Expose the plate to iodine vapour for 5 mino Reference solution Introduce 2 mL of tin standard solmion
Examine the chromatogram and locate the band (5 ppm (Sn) R) into a 50 mL flask containing 5 mL of a
corresponding to plastic additives 04 and 05 (R p = O). 20 per cent V/V solution of sulfuric acid R and dilute ro
Remove the are a of silica gel corresponding to this zone. 50 mL with water R irnmediately before use .
Similarly remove a corresponding area of silica gel as a blank Wavelength Use the emission oftin at 189.99 nm, the
reference. Separately shake both samples for 15 min with spectral background being taken at 190.10 nm.
40 rnL of methanol R . Filter, rinse with 2 quantities, each of Verify the absence of tin in the hydrochloric acid used.
10 mL of methanol R, add the rinsings to the filtrate and
Zinc Maximum 0.2 per cent.
evapora te to dryness. The difference between the masses of
both residues is not more than 10 mg. Atomic absorption spectrometry (2.2.23, M echod ¡) .
Plastic additive 03 Infrared absorption spectrophotometry Test solution Dilute solution SI 100 times with 0.1 M
(2.2.24). hydrochloric acid.
Preparation Wash precipitate B2 obtained during the Reference solutions Prepare the reference solutions using
identificatipn and contained in the tared sintered-glass filter zinc standard solution (J 00 ppm Zn) R, diluting with 0.1 M
(40) (2. 1.2) with anhydrous ethanol R . Dry to constant mass hydrochlOlic acid.
over diphosphonls penlOxide R and weigh the filter. Source Zinc hollow-cathode lampo
The residue weighs not more than 20 mg. Wavelength 213.9 nm.
Comparison Plastic additive 03 CRS. Atomisation device Air-acetylene flameo
Barium Maximum 5 ppm. Verify the absence of zinc in the hydrochloric acid used.
Heavy metals (2.4.8) Maximum 50 ppm.
2014 Appendix XX B V-A557
To 10 mL of solution SI add 0.5 mL of phenolphthalein - 2,2' -bis( octadecyloxy)-5,5' -spirobi[1,3,2-

solution R and then strong sodium hydroxide solution R until a dioxaphosphinane] (plastic additive 14) : maximum
pale pink colour is obtained. Dilute to 25 mL with water R. 0.3 per cent;
12 mL of solution complies with test A. Prepare the - didodecyl 3,3' -thiodipropionate (plastic additive 16):
reference solution using lead standard solution (2 ppm Pb) R. maximum 0.3 per cent;
Water extractable substances Maximum 0.3 per cent. - dioctadecyI3,3'-thiodipropionate (plastic additive 17):
Evaporate 50.0 mL of solution S2 to dryness on a water-bath maximum 0.3 per cent;
and dry at 100-105 oC until constant mass. Carry out a - tris[2,4-bis(1,1-dimethylethyl)phenyl] phosphite (plastic
blank titration with 50.0 mL of water jor injections R. additive 12): maximum 0.3 per cent;
The residue weighs not more than 7.5 mg taking into - plastic additive 18: maximum 0.1 per cent;
account the blank test.
- copolymer of dimethyl succinate and (4-hydroxy-2,2,6,6-
Assay tetramethylpiperidin-1-yl)ethanol (plastic additive 22) :
Carry out the oxygen-flask method (2.5.10) using 50.0 mg. maximum 0.3 per cent.
Absorb the combustion products in 20 mL of 1 M sodium The total of antioxidant additives listed aboye do es not
hydroxide. To the solution obtained add 1 mL of dibutyl exceed 0.3 per cent.
phthalate R, 2.5 mL of nitric acid R, 5 mL ofjerric ammonium
- hydrotaleite: maximum 0.5 per cent;
sulfate solurion R2 and 10.0 mL of 0.1 M szlver nitrate. Titrate
with 0.05 M ammonium thiocyanate until a reddish-yellow - alkanamides: maximum 0.5 per cent;
colour is obtained. Carry out a blank test. - alkenamides: maximum 0.5 per cent;
1 mL of 0.1 M silver nitrate is equivalent to 6.25 mg of - sodium silico-aluminate: maximum 0.5 per cent;
poly(vinyl chloride) . - silica: maximum 0.5 per cent;
- sodium benzoate: maximum 0.5 per cent;
- fatry acid esters or salts: maximum 0.5 per cent;
- trisodium phosphate: maximum 0.5 per cent;
B. Polyolefines
-liquid paraffin: maximum 0.5 per cent;
- zinc oxide: maximum 0.5 per cent;
Definition - tale: maximum 0.5 per cent;
Polyolefines are obtained by polymerisation of ethylene or - magnesium oxide: maximum 0.2 per cent;
propylene or by copolymerisation of these substances with - caleium stearate or zinc stearate or a mixture of both:
not more than 25 per cent of higher homologues (C 4 to CIO) maximum 0.5 per cent;
or of carboxylic acids or of esters. Certain materials may be - titanium dioxide: maximum 4 per cent.
mixtures of polyolefines. '
Production that the qualitative and quantitative composition of the type
A certain number of additives are added to the polymer in sample is satisfactory for each production batch.
order to optimise their chemical, physical and mechanical Characters
properties in order to adapt them for the intended use. All of Appearance Powder, beads, granules or, after
these additives are chosen from the appended list which transformation, sheets of varying thickness or containers.
specifies for each product the maximum allowable content.
Solubility Practically insoluble in water, soluble in hot
They may contain at most 3 antioxídants, 1 or several aromatic hydrocarbons, practically insoluble in anhydrous
lubricants or antiblocking agents as well as titanium dioxide
ethanol, in hexane and in methano!.
as an opacifYing agent when the material must provide
protection from light. They soften at temperatures between 65 oC and 165 oc.
They burn with a blue flameo
- burylhydroxytoluene (plastic additive 07): maximum
0.125 per cent; 1dentification
- pentaerythriryl tetrakis [3-(3,5-di-tert-butyl-4- lf necessary, cut the samples oj the material to be examined into
hydroxyphenyl)propionate] (plastic additive 09): pie ces oj maximum dimension on a side oj not greater than 1 cm.
maximum 0.3 per cent; A. Infrared absorption spectrophotometry (2.2.24) .
- 1,3,5-tris(3,5-di-tert-buryl-4-hydroxybenzyl)-S-triazine- Preparation To 0.25 g add 10 mL of toluene R and boil
2,4,6(lH,3H,5H)-trione, (plastic additive 13): maximum under a refiux condenser for about 15 min; place a few
0.3 per cent; drops of the solution obtained on a sodium chloride slide
- octadecyl 3-(3,5-di~tert-butyl-4-hydroxyphenyl)propionate and evaporate the solvent in an oven at 80 oC .
(plastic additive 11): maximum 0.3 per cent; Absorption maxima At 2920 cm- l, 2850 cm- l ,
- ethylene bis [3,3-bis [3-( 1, 1-dimethylethyl)-4- 1475 cm- l , 1465 cm- l , 1380 cm- l , 1170 cm- \ 735 cm- l
hydroxyphenyl]butanoate] (plastic additive 08): maximum and 720 cm-l.
0.3 per cent; The spectrum obtained is identical to the spectrum obtained
- dioctadecyl disulfide (plastic additive 15): maximum with the material selected for the type sample. If the material
0.3 per cent; to be examined is in the form of sheets, the identification
- 4,4',4 ff -(2,4,6-trimethylbenzene-l ,3,5- may be determined directly on a cut pie ce of suitable size.
triyltrismethylene)tris [2,6-bis(I, 1-dimethylethyl)phenol] B. It complies with the supplementary tests corresponding
(plastic additive 10): maximum 0.3 per cent; to the additives presento
V-A558 Appendix xx B 2014
e. In a platinum crucible, mix about 20 mg with 1 g of Extractable aluminium M aximum 1 ppm.

potassium hydrogen sulfate R and heat until completely Inductively coupled plasma-atomic emission spectrometry
melted. Allow to cool and add 20 mL of di/ute sulfuric (2.2.57) .
acid R. Heat gently. Filter the resulting solution. To the Test solution Use solution S3.
filtrate add 1 mL of phosphoric acid R and 1 mL of stTOng
hydTOgen peroxide solution R. If the substance is opacified
aluminium standard solution (200 ppm Al) R, diluting with
with titanium dioxide, an orange-yellow colour develops.
O. 1 M hydrochloric acid.
Tests Wavelength Use the emission ofaluminium at 396.15 nm,
Jf necessary, cut the samples of the material lo be examined inlO the spectral background being taken as 396.25 nm.
pieces of maximuin dimension on a side of not greater than 1 cm. Verify the absence of aluminium in the hydrochloric acid
Solution SI Use solution SI within 4 h of preparation. Place used.
25 g in a borosilicate-glass flask with a ground-glass neck. Extractable titanium Maximum 1 ppm.
Add 500 mL of water for injections R and boil under a reflux
condenser for 5 h. Allow to cool and decanto Reserve a
(2.2. 57).
portion of the solution for the test for appearance of solution
SI and filter the rest through a sintered-glass filter (16) Test solution Use solution S3 .
(2.1.2). Reference solutions Prepare the reference solutions using
Solution S2 Place 2.0 g in a conical borosilicate-glass flask titanium standard solution (100 ppm Ti) R, diluting with
with a ground-glass neck. Add 80 mL of lOluene R and boil 0.1 M hydrochloric acid.
under a reflux condenser with constant stirring for 90 mino Wavelength Use the emission of titanium at 336.12 nm,
Allow to cool to 60 oC and add with continl!ed stirring the spectral background being taken as 336.16 nm.
120 mL of methanol R. Filter the solution through a Verify the absence of titanium in the hydrochloric acid used .
sintered-glass filter (16) (2. 1.2). Rinse the flask and the
Extractable zinc Maximum 1 ppm.
filter with 25 mL of a mixture of 40 mL of lOluene R and
60 mL of methanol R, add the rinsings to the filtrate and Atomic absorption spectrometry (2.2.23, Method J).
dilute to 250 mL with the same mixture of solvents . Test solution Use solution S3.
Prepare a blank solution. Reference solutions Prepare the reference solutions using
Solution S3 Place 100 g in a conical borosilicate-glass zinc standard solution (JO ppm Zn) R, diluting with 0.1 M
flask with a ground-glass neck. Add 250 mL of 0.1 M hydrochloric acid.
hydrochloric.acid and boil under a reflux condenser with Source Zinc hollow-cathode lamp.
constant stirring for 1 h . Allow to cool and decant the Wavelength 2 13 .9 nm.
solution.
Atomisation device Air-acety1ene flame.
Appearance of solution SI Solution SI is clear (2.2.1)
Verify the absence of zinc in the hydrochloric acid used.
and colourless (2.2.2, M ethod lI) .
Extractable heavy metals (2.4.8) Maximum 2.5 ppm.
Acidity or alkalinity To 100 mL of solution SI, add
0.15 mL of BRP indicalOr solution R. Not more than 1.5 mL Evaporate 50 mL of solution S3 to about 5 mL on a water-
of 0.01 M sodium hydroxide is required to change the colour bath and dilute to 20.0 mL with water R . 12 mL of the
of the indicator to blue. To 100 mL of solution SI add solution complies with test A. Prepare the reference solution
0.2 mL of methyl orange solution R. Not more than 1 mL of using 2.5 mL of lead standard solution (JO ppm Pb) R.
O. 01 M hydrochloric acid is required to initiate the colour SuIfated ash (2.4.14) Maxirnum 1.0 per cent, deterrnined
change of the indicator from yellow to orange. on 5.0 g. This limit do es not apply to material that has been
Absorbance (2.2.25) Maximum 0.2, determined between opacified with titanium dioxide.
wavelengths of 220 nm and 340 nm on solution SI. SUPPLEMENT ARY TESTS
Reducing substances T o 20 mL of solution SI add 1 mL These tests are lO be camed out, in whole or in part, only if
of dilute sulfuric acid R and 20 mL of 0.002 M potassium required by the stated composition or the use of the material.
permanganate. Boil under a reflux condenser for 3 min and Phenolic antioxidants Liquid chromatography (2.2.29).
cool immediately. Add 1 g of potassium iodide R and titrate Solvent mixture acelOnitrile R, tetrahydrofuran R (50:50
iinmediately with 0.01 M sodium thiosulfate, using 0.25 mL of V/V).
starch solution R as indicator. Carry out a blank titration.
Test solution S21 Evaporate 50 mL of solution S2 to
The difference between the titration volumes is not more
dryness in vacuo at 45 oC. Dissolve the residue in 5.0 mL of
than 3.0 mL.
the solvent mixture. Prepare a blank solution from the blank
Substances soluble in hexane Place 10 g in a 250 mL solution corresponding to solution S2 .
conical borosilicate-glass flask with a ground-glass neck.
Add 100' mL of hexane R and boil under a reflux condenser
dryness in vacuo at 45 oc. Dissolve the residue with 5.0 mL
for 4 h, stirring constantly. Cool in iced water and filter
of methylene chloride R. Prepare a blank solution from the
rapidly (the filtration time must be les s than 5 mini
blank solution corresponding to solution S2.
if necessary the filtration may be accelerated by applying
pressure to the solution) through a sintered-glass filter (16) Test solution S23 Evaporate 50 mL of solution S2 to
(2. 1.2) maintaining the solution at about O 0e. Evaporate dryness in vacuo at 45 oC . Dissolve the residue in 5.0 mL of
20 mL of the filtrate in a tared borosilicate-glass dish on a a mixture of equal volumes of acelOnitrile R and a 10 gIL
water-bath. Dry the residue in an oven at 100-105 oC for solution of tert-butylhydroperoxide R in tetrahydrofuran R.
1 h. The m ass of the residue obtained must be within Close the flask and allow to stand for 1 h. Prepare a blank
10 per cent of that of the residue obtained with the type solution using the blank of solution S2.
sample and does not exceed 5 per cent.
2014 Appendix XX B V-A559
Of the following referenee solutions, prepare only lhose that are - the chromatogram obtained with test solution S21 only
neeessary for the analysis of the phenolie antioxidants stated in the show peaks due to antioxidants stated in the composition
composition of lhe substanee to be examined. and minor peaks that also appear in the chromatogram
Referenee solution (a) Dissolve 25.0 mg of corresponding 10 the blank solution.
butylhydroxytoluene CRS (plastic additive 07) and 60.0 mg of Limit The areas of the peaks in the chromatogram
plastie additive 08 CRS in 10.0 mL of the solvent mixture. obtained with test solution S21 are less than the
Dilute 2.0 mL of this solution to 50.0 mL with the solvent corresponding areas of the peaks in the chromatograms
mixture. obtained with reference solutions (d) and/or (e).
Referenee solution (b) Dissolve 60.0 mg of plastie additive B. If the substance to be examined contains one or more of
09 CRS and 60.0 mg ofplastie additive 10 CRS in 10.0 mL the following antioxidants:
of the solvent mixture. Dilute 2.0 mL of this solution to - plastic additive 09;
50.0 mL with the solvent mixture. - plastic additive 10;
Reference solution (e) Dissolve 60.0 mg of plastic additive - plastic additive 11;
11 CRS and 60.0 mg of plastie additive 12 CRS in 10.0 mL
of methylene ehloride R . Dilute 2.0 mL of this solution to - plastic additive 12;
50.0 mL with methylene ehloride R . - plastic additive 13;
Reference solution (d) Dissolve 25.0 mg of plastie carry out the test as described aboye with the following
additive 07 CRS in 10.0 mL of the solvent mixture. Dilute modifications .
2.0 mL of this solution to 50.0 mL with the solvent mixture. Mobile phase water R , tetrahydrofuran R, aeetonitrile R
Referenee solution (e) Dissolve 60 .0 mg of plastie additive (10:30:60 VIV/V).
08 CRS in 10.0 mL of the solvent mixture. Dilute 2.0 mL of Flow rate 1.5 mUmin.
this solution to 50.0 mL with the solvent mixture. Injeetion 20 J.lL of the test solution S21, the
Referenee solution (f) Dissolve 60.0 mg of plastie additive corresponding blank solution, the reference solution (b) and
13 CRS in 10.0 mL of the solvent mixture . Dilute 2.0 mL of the reference solutions of the antioxidants on the list aboye
this solution 10 50.0 mL with the solvent mixture. that are stated in the composition.
Referenee solution (g) Dissolve 60.0 mg of plastie additive System suitability:
09 CRS in 10.O mL of the solvent mixture. Dil ute 2. O mL of - resolution: minimum 2.0 between the peaks due 10 plastic
this solution 10 50.0 mL with the solvent mixture. additive 09 and plastic additive 10 in the chromatogram
Referenee, solution (h) Dissolve 60.0 mg of plastie obtained with reference solution (b);
additive 10 CRS in 10.0 mL ofthe solvent mixture. Dilute - the chromatogram obtained with test solution S21 only
2.0 mL of this solution to 50.0 mL with the solvent mixture. show peaks due to antioxidants stated in the composition
Referenee solution (i) Dissolve 60.0 mg of plaslic additive and minor peaks that also appear in the chromatogram
11 CRS in 10.0 mL of methylene ehloride R . Dilute 2.0 mL of corresponding to the blank solution.
this solution 10 50.0 mL with methylene ehloride R. Limit The areas of the peaks in the chromatogram
Referenee solution (j) Dissolve 60.0 mg of plaslie additive obtained with test solution S21 are less than the
12 CRS in 10.0 mL of methylene ehloride R . Dilute 2.0 mL of corresponding areas of the peaks in the chromatograms
this solution 10 50.0 mL with methylene ehloride R. obtained with reference solutions of the antioxidants on the
Referenee solution (k) Dissolve 20.0 mg of plastie list aboye that are stated in the composition.
additive 18 CRS in 10.0 mL of a mixture of equal volumes C. If the substance to be examined contains plastic additive
of aeetonitrile R and a 10 gIL solution of 11 and/or plastic additive 12, carry out the test as
tert-butylhydroperoxide R in tetrahydrofuran R . Allow to stand described for plastic additive 07 and/or plastic additive
in a closed container for 1 h. Dilute 2.0 mL of this solution 08 with the following modifications.
to 50.0 mL with the solvent mixture. Mobile phase water R, 2-propanol R , methanol R (5:45:50
A. If the substance to be examined contains plastic additive VIV/V).
07 ami/or plastic additive 08, carry out the test as Flow rate 1.5 mUmin.
follows. Injeetion 20 J.lL of the test solution S22, the
Column: corresponding blank solution, the reference solution (e), and
- size: 1 = 0.25 m, 0 = 4.6 mm; either the reference solution (i) or (j) or the reference
- stationary phase: octadeeylsilyl szliea gel for ehromatography R solutions (i) and (j).
(5 J.lm). System suitability:
Mobile phase water R; aeetonitrile R (30:70 V/V). - resolution: minimum 2.0 between the peaks due 10 plastic
Flow rate 2 mUmin. additive 11 and plastic additive 12 in the chromatogram
Deteetion Spectrophotometer at 280 nm.
obtained with reference solution (e);
Injeetibn 20 J.lL of the test solution S21, the
- the chromatogram obtained with test solution S22 only
corresponding blank solution, the reference solution (a), and show peaks due 10 antioxidants stated in the composition
either the reference solutions (d) or (e) or the reference and minor peaks that also appear in the chromatogram
solutions (d) and (e). corresponding 10 the blank solution.
Limit The areas of the peaks in the chromatogram
Run time 30 mino
obtained with test solution S22 are les s than the
System suitability: corresponding areas of the peaks in the chromatograms
- resolution: minimum 8.0 between the peaks due to plastic obtained with reference solutions (i) and/or (j) .
additive 07 and plastic additive 08 in the chromatogram D. If the substance to be examined contains plastic additive
obtained with reference solution (a); 18, carry out the test as described for plastic additive 07
V-A560 Appendix xx B 2014
and/or plastic additive 08 with the fol!owing corresponding positions in the chromatograms obtained with
modifications. the reference solutions.
Mobile phase tetrahydrofuran R, aeetonitrile R (20:80 VIV). Plastic additive 22 Liquid chromatography (2.2.29) .
Flow rate 1.5 mUmin. Test solution Evaporate 25 mL of solution S2 lO dryness
Detection SpectropholOmeter at 270 nm. in vaeuo at 45 oC. Dissolve the residue in 10 mL of toluene R
and 10 mL of a 10 gIL solution of tetrabutylammonium
Injection 20 ¡.¡L of the test solution S23, the
hydroxide R in a mixture of 35 volumes of toluene R and
corresponding blank solution and the reference solution (k).
65 volumes of anhydrous ethanol R. Boil under a refiux
System suitability: condenser for 3 h. Allow lO cool and filter if necessary.
- resolution: minimum 6.0 between the 2 principal peaks Reference solution Dissolve 30 mg of plastie additive
(approximate retention times of 3.5 and 5.8) in the 22 CRS in 50 mL of toluene R. Add 1 mL of this solution to
chromatogram obtained with reference solution (k); 25 mL of blank solution S2 and evaporate to dryness in
- the chromalOgram obtained with test solution S23 only vaeuo at 45 oc. Dissolve the residue in 10 mL of toluene R
show peaks due to antioxidants stated in the composition and 10 mL of a 10 gIL solution of tetrabutylammoniUln
and minor peaks that also appear in the chromatogram hydroxide R in a mixture of 35 volumes of toluene R and
corresponding lO the blank solution. 65 volumes of anhydrous ethanol R. Boil under a refiux
Limit The areas of the peaks in the chromatogram condenser for 3 h. Allow lO cool and filter if necessary.
obtained with test solution S23 are les s than the Column:
corresponding areas of the peaks in the chromatograms - size: 1 = 0.25 m, (2) = 4.6 mm;
obtained with reference solution (k).
- stationary phase: aminopropylsilyl silica gel for
Non-phenolic antioxidants Thin-Iayer chromatography ehromatography R (5 ¡.¡m).
(2.2.27).
Mobile phase anhydrous ethanol R, hexane R (11:89
. Test solution S24 Evaporate 100 mL of solution S2 lO VN).
dryness in vaeuo at 45 oC. Dissolve the residue in 2 mL of
acidified methylene ehloride R. Flow rate 2 mUmin.
Detection spectrophotometer at 227 nm.
Reference solution (1) Dissolve 60 mg of plastie additive
14 CRS in 10 mL of methylene ehloride R. Dilute 2 mL of Injection 20 ¡.¡L.
this solution lO 1O mL with acidified methylene ehloride R. Run time 10 mino
Reference solution (m) Dissolve 60 mg of plastie additive System suitability:
15 CRS in 10 mL of methylene ehloride R. Dilute 2 mL of - resolution: minimum 7 between the peaks due lO the
this solution to 10 mL with acidified methylene ehloride R. "diol" component and lO the diluent of the reference
Reference solution (n) Dissolve 60 mg of plastie additive solution.
16 CRS in 10 mL of methylene ehloride R. Dilute 2 mL of Limit The area of the peak due to the "diol" component
this solution lO 10 mL with acidified methylene ehloride R. from plastic additive 22 in the chromalOgram obtained with
Reference solution (o) Dissolve 60 mg of plastie additive the test solution is less than the corresponding peak in the
17 CRS in 10 mL of methylene ehloride R. Dilute 2 mL of chromatogram obtained with the reference solution.
this solution lO 10 mL with aeidified methylene ehloride R. Amides and stearates Thin-Iayer chromatography
Reference solution (p) Dissolve 60 mg of plastie additive (2.2.27).
16 CRS and 60 mg of plastie additive 17 CRS in 10 mL of Test solution Use test solution S24 described in the test
methylene ehloride R. Dilute 2 mL of this solution lO 10 mL for non-phenolic antioxidants.
with acidified methylene ehloride R.
Reference solution (q) Dissolve 20 mg of stearic acid
Plate TLC siliea gel GF254 plate R. (plaslic additive 19 CRS) in 10 mL of methylene chloride R.
Jtfobile phase A hexane R. Reference solution (r) Dissolve 40 mg of oleamide
Mobile phase B methylene ehloride R. (plastie additive 20 CRS) in 20 mL of methylene ehloride R.
Application 20 ~lL of the test solution S24, the reference Reference solution (s) Dissolve 40 mg of erucamide
. solution (P) and the reference solutions corresponding to al! (plastie additive 21 CRS) in 20 mL of methylene ehloride R .
the phenolic and non-phenolic antioxidants mentioned in the Plate TLC siliea gel GF254 place R (2 plates).
type composition of the material to be examined.
A. Mobile phase anhydrous ethanol R, trimethylpentane R
Development A Over a path of 18 cm with mobile phase (25:75 VIV).
A.
Application 10 ¡.¡L of the test solution S24 and reference
Drying A In airo solution (q).
Development B Over a path of 17 cm with mobile Development Over a path of 10 cm.
phase B.
Drying In air.
Drying B In airo
Detection Spray with a 2 gIL solution of
Detection Examine in ultraviolet light at 254 nm; spray diehlorophenolindophenol sodium salt R in anhydrous ethanol R
with aleoholie iodine solution R and examine in ultraviolet light and heat in an oven at 120 oC for a few minutes lO intensifY
at 254 nm after 10-15 mino the spots.
System suitability Reference solution (p): Limit Any spot corresponding to plastic additive 19 in the
- the chromatogram shows 2 clearly separated spots. chromatogram obtained with test solution S24 is identical in
Limit Any spots in the chromatogram obtained with test position to (R p = about 0.5) but not more intense than the
solution S24 are not more intense than the spots in the spot in the chromalOgram obtained with reference
solution (q) .
2014 Appendix XX e V-A561
B. Mobile phase A hexane R. Tests

Mobile phase B methanol R, methylene chloride R (5:95 1f necessaly, cut the samples of the material to be examined into
V/V). pieces of maximum di11'lension on a side of not greater than 1 cm.
Application 10 llL of the test solution S24 and the Solution S1 Place 25 g in a borosilicate-glass flask with a
reference solutions (r) and (s). ground-glass neck. Add 500 mL of water fol' injectio11S R and
Developrnent A Over a path of 13 cm with mobile phase heat under a reflux condenser for 5 h. Allow to cool and
A. decanto Keep part of the solution for the test for appearance
Drying A In air. of solution. Filter the rest through a sintered glass filter (16)
(2. 1.2). Use solution SI within 4 h ofpreparation.
Developrnent B Over a path of 10 cm with mobile
Solution S2 Place 2.0 g in a conical borosilicate-glass flask
phase B.
with a ground-glass neck. Add 80 mL of toluene R and boil
Drying B In air. under a reflux condenser with constant stirring for 1 h
Detection Spray with a 40 giL solution of phosphomolybdic 30 mino AlIow to cool to 60 oC and add with continued
acid R in anhydrous ethanol R . Heat in an oven at 120 oC stirring 120 mL of methanol R. Filter the solution through a
until spots appear. sintered-glass filter (16) (2. 1.2). Rinse the flask and the filter
Lirnit Any spots. corresponding 10 plastic additive 20 or with 25 mL of a mixture of 40 mL of toluene R and 60 mL
plastic additive 21 in the chromatogram obtained with test of methanol R, add the rinsings 10 the filtrate and dilute 10
solution S24 are identical in position 10 (R p = about 0.2) 250 mL with the same mixture of solvents. Prepare a blank
but not more intense than the corresponding spots in the solution.
chromatograms obtained with reference solutions (r) and (s). Solution S3 Place 100 g in a conical borosilicate-glass
flask with a ground-glass neck. Add 250 mL of 0.1 M
hydrochloric acid and boil under a reflux condenser with
constant stirring for 1 h. AIlow 10 cool and decant the
C. Polyethylene solution.
Appearance ofsolution Solution SI is clear (2.2.1) and
1. Polyethylene Without Additives for Containers for
colourless (2.2.2, M ethod 11).
Parenteral Preparations and for Ophthalmic
Preparations Acidity or aIkalinity To 100 mL of solution SI add
0.15 mL of BRP indicator solution R. Not more than 1.5 mL
(Ph. Eur. method 3.1.4) of O. O1 M sodiUln hydroxide is required to change the colour
of the indicator 10 blue. To 100 mL of solution SI add
Definition
0.2 mL of methyl orange sohaion R. Not more than 1.0 mL of
Polyethylene without additives is obtained by the 0.01 M hydrochloric acid is required 10 reach the beginning of
polymerisation of ethylene under high pressure in the the colour change of the indicator from yellow 10 orange.
presence of oxygen or free-radical-forming initiators as
Absorbance (2.2.25) Maximum 0.2, determined between
catalyst.
wavelengths of 220 nm and 340 nm on solution SI.
Characters Reducing substances To 20 mL of solution SI add 1 mL
of dilute sulfuric acid R and 20 mL of 0.002 M potassiu11'l
Appearance Beads, granules, powder or, after
pemwnganate. Boil under a reflux condenser for 3 min and
transformation, translucent sheets of varying thickness or
cool immediately. Add I g of potassiwn iodide R and ti trate
containers.
Solubility Practically insoluble in water, soluble in hot starch solution R as indicator. Carry out a blank titration.
aroma tic hydrocarbons, practically insoluble in anhydrous The difference between the titration volumes is not more
ethanol, in hexane and in methanol. than 0.5 mL.
It softens at temperatures beginning at 65 oc. Substances soluble in hexane Place 10 g in a 250 mL
Relative density 0.910 to 0.937 . conical borosilicate-glass ftask with a ground-glass neck.
Add 100 mL of hexane R and boil under a reflux condenser
Identification for 4 h, stirring constantly. Cool in iced water and filter
1f necessary, cut the ~amples of the material to be examined into rapidly through a sintered-glass filter (16) (2.1.2) maimaining
pieces of maximum dimension on a side of not greater than 1 cm. the solution at O oC (the filtration time must be less than
A. Infrared absorption spectrophotometry (2.2.24). 5 min; if necessary the filtration may be accelerated by
applying pressure 10 the solution). Evaporate 20 mL of the
Preparation To 0.25 g add 10 mL of toluene R and
filtra te in a tared glass dish on a water-bath. Dry the residue
boil under a reflux condenser for about 15 mino Place a
in an oven at 100-105 oC for 1 h. The mas s of the residue
few drops of the solution on a sodium chloride disc and
obtained is within 10 per cent of the residue obtained with
evaporate the solvent in an oven at 80 oc.
the type sample and does not exceed 5 per cent.
Absorption rnaxirna At 2920 cm- 1, 2850 cm- 1 ,
Additives Thin-Iayer chromatography (2.2.27).
1465 cm- 1, 730 cm- 1 and 720 cm- l.
Test solution Evaporate 50 mL of solution S2 to dryness
The spectrum obtained is identical to that obtained with
in vacuo at 45 oc. Dissolve the evaporation residue with
the material selected for the type sample. If the material
5 mL of methylene chloride R . Prepare a blank solution from
10 be examined is in the form of sheets, the
the blank solution corresponding 10 solution S2.
identification may be performed directly on a cut piece
of suitable size. Reference solution Dissolve 20 mg of plastic additive
15 CRS and 20 mg of plastic additive 08 CRS in methylene
B. Additives (see Tests).
chloride R and dilute to 10 mL with the same solvent.
Plate TLC silica gel G plate R .
V-A562 Appendix xx e 2014
Mobile phase A hexane R. - ethylene bis [3,3-bis [3-( 1, l-dimethylethyl)-4-

Mobile phase B methanol R, methylene chloride R (5:95 hydroxyphenyl)butanoate) (plastic additive 08): maximum
V/V) . 0.3 per cent;
Application 1o ~L. - dioctadecyl disulfide (plastic additive 15): maximum
0.3 per cent;
Development A Over a path of 13 cm using mobile
phase A. - 4,4' ,4"-(2,4,6-trimethylbenzene-l ,3,5-
triyltrismethylene )tris [2,6-bis (1, l-dimethylethyl)phenol)
Drying A In airo
(plastic additive 10): maximum 0.3 per cent;
Development B Over a path of 10 cm using mobile
- 2,2'-bis(octadecyloxy)-5,5 '-spirobi[I,3,2-
phase B.
dioxaphosphinane) (plastic additive 14): maximum
Drying B In air. 0.3 per cent;
Detection Spray with a 40 giL solution of phosphomolybdic - didodecyl 3,3'-thiodipropionate (plastic additive 16):
acid R in ethanol (96 per cem) R and heat at 120 oC until the maximum 0.3 per cent;
spots appear in the chromatogram obtained with the
- dioctadecyl 3,3'-thiodipropionate (plastic additive 17):
reference solution.
maximum 0.3 per cent;
System suitability Reference solution:
- tris [2,4-bis( 1, l-dimethylethyl)phenyl) phosphite (plastic
- the chromatogram shows 2 separated spots. additive 12): maximum 0.3 per cent.
Limit No spot appears in the chromatogram obtained with The total of antioxidant additives listed aboye does not
the test solution, except for a spot which may be at the exceed 0.3 per cent.
solvent front from the first development and which
- hydrotalcite: maximum 0.5 per cent;
corresponds to oligomers. Disregard any spots corresponding
to those obtained in the chromatogram with the blank - alkanamides: maximum 0.5 per cent;
solution. - alkenamides: maximum 0.5 per cent;
Extractable heavy metals (2.4.8) Maximum 2.5 ppm. - sodium silico-aluminate: maximum 0.5 per cent;
Evaporate 50 mL of solution S3 to about 5 mL on a - silica: maximum 0.5 per cent;
water-bath and dilute to 20 mL with water R. 12 mL of - sodium benzoate: maximum 0.5 per cent;
solution complies with test A. Prepare the reference solution - fatty acid esters or salts: maximum 0.5 per cent;
using 2.5 mL of lead standard solution (lO ppm Pb) R.
- trisodium phosphate: maximum 0.5 per cent;
Sulfated ash (2.4.14) Maximum 0.02 per cent,
determined on 5.0 g. -liquid paraffin: maximum 0.5 per cent;
2. Polyethylene With Additives for Containers for - magnesium oxide: maximum 0.2 per cent;
Parenteral Preparations and for Opthalmic - calcium stearate or zinc stearate or a mixture of both:
Preparations maximumO.5 per cent;
(Ph. Eur. method 3.1.5) - titanium dioxide only for materials for containers for
ophthalmic use: maximum 4 per cent.
Definition
Polyethylene with additives is obtained by the polyrnerisation that the qualitative and quantitative composition of the type
of ethylene under pressure in the presence of a catalyst or by sample is satisfactory for each production batch.
copolyrnerisation of ethylene with not more than 25 per cent
of higher alkene homologues (C 3 to C IO). Characters
Appearance Powder, beads, granules or, after
Production
transformation, translucent sheets of varying thicknesses or
A certain number of additives are added to the polyrner in containers.
order to optimise their chemical, physical and mechanical
Solubility Practically insoluble in water, soluble in hot
properties in order to adapt them for the intended use.
aroma tic hydrocarbons, practically insoluble in anhydrous
AlI these additives are chosen from the appended list which
ethanol, in hexane and in methanol.
specifies for each product the maximum allowable contento
It softens at temperatures between 70 oC and 140 oc.
They may cdntain at most 3 antioxidants, 1 or several
lubricants or antiblocking agents as well as titanium dioxide Relative density 0.890 to 0.965.
as an opacifying agent wHen the material must provide 1dentification
protection from light. lf necessary, cut the samples 01 the material LO be examined inLO
- butylhydroxytoluene (plastic additive 07): maximum pie ces 01 maximum dimension on a side 01 not greater than 1 cm.
0.125 per cent; A. Infrared absorption spectrophotometry (2.2.24).
- pentaerythrityl tetrakis [3- (3, 5-di-tert-butyl-4- Preparation To 0.25 g add 10 mL of LOluene R and boil
hydroxyphenyl)propionate) (plastic additive 09) : under a reflux condenser for about 15 mino Place a few
maximum 0.3 per cent; drops of the solution on a sodium chloride disc and
- 1,3,5-tris (3,5-di-tert-butyl-4-hydroxybenzyl)cS- evaporate the solvent in an oven at 80 oC.
triazine2,4,6(IH,3H,5H)-trione (plastic additive 13): Absorption maxima At 2920 cm- 1, 2850 cm- l ,
maximum 0.3 per cent; 1465 cm- \ 1375 cm- 1, 1170 cm-l, 730 cm- l and
- octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, 720 cm - l .
(plastic additive 11): maximum 0.3 per cent; The spectrum obtained is identical to the spectrum obtained
with the material selected for the type sample. If the material
to be examined is in the form of sheets, the identification of the residue obtained must be within 10 per cent of the
may be performed directly on a cut piece of suitable size. residue obtained with the type sample and does not exceed
B. It complies with the supplementary tests corresponding 5 per cent.
to the additives present (se e Tests). Extractable aluminium Maximum 1 ppm.
C. In a platinum crucible, mix about 20 mg with 1 g of Inductively coupled plasma-atomic emission spectrometry
potassium hydrogen sulfate R and heat until completely (2.2.57).
melted. Allow to cool and add 20 mL of dilute sulfuric Test solution Use solution S3.
acid R. Heat gently. Filter the resulting solution. To the
filtrate add 1 mL of phosphoric acid R and 1 mL of strong aluminium standard solution (200 ppm AV R, diluting with
hydrogen peroxide solurion R. If the substance is opacified 0.1 M hydrochloric acid.
with titanium dioxide, an orange-yellow colour develops.
Wavelength Use the emission of aluminium at 396.15 nm,
Tests the spectral background being taken as 396.25 nm.
1f necessary, cut the samples of the material lO be examined inlO Verify the absence of aluminium in the hydrochloric acid
pieces of maximum dimension on a side of not greater than 1 cm. used.
Solution SI Place 25 g in a borosilicate-glass ftask with a Extractable chromium Maximum 0.05 ppm.
ground-glass neck. Add 500 mL of water for injections R and Inductively coupled plasma-atomic emission spectrometry
boil under a reftux condenser for 5 h. Allow to cool and (2.2.57).
decanto Reserve a portion of the solution for the test for
appearance of solution and filter the rest through a sintered- Test solution Use solution S3.
glass filter (16) (2. 1.2) . Use within 4 h ofpreparation. Reference solutions Prepare the reference solutions using
chromium standard solution (lOO ppm Cr) R, diluting with a
Solution S2 Place 2.0 g in a conical borosilicate-glass ftask
with a ground-glass neck. Add 80 mL of lOluene R and boil mixture of 2 volumes of hydrochloric acid R and 8 volumes of
water R .
under a reftux condenser with constant stirring for 90 mino
Allow to cool to 60 oC and add with continued stirring Wavelength Use the emission of chromium at 205.55 nm,
120 mL of methanol R. Filter the solution through a sintered- the spectral background being taken as 205.50 nm.
glass filter (16) (2.1.2). Rinse the ftask and the filter with Verify the absence of chromium in the hydrochloric acid
25 mL of a mixture of 40 mL of lOluene R and 60 mL of used.
methanol R, add the rinsings to the filtrate and dilute to Extractable titanium Maximum 1 ppm.
250.0 mL with the same mixture of solvents. Prepare a blank
solution. (2.2.57).
Solution S3 Place 100 g in a conical borosilicate-glass
Test solution Use solution S3.
ftask with a ground-glass neck. Add 250 mL of 0. 1 M
hydrochloric acid and boil under a reftux condenser with
titanium standard solution (lOO ppm Ti) R, diluting with
constant stirring for 1 h . Allow to cool and decant the
solution. 0.1 M hydrochloric acid.
Appearance of solution Solution SI is c1ear (2.2.1) and Wavelength Use the emission of titanium at 336.12 nm,
colourless (2.2.2, Method 11) . the spectral background being taken as 336.16 nm.
Acidity or aIkalinity To 100 mL of solution SI add Verify the absence of titanium in the hydrochloric acid used.
0.15 mL of BRP indicalOr solution R. Not more than 1.5 mL Extractable vanadium Maximum 0.1 ppm.
of O. 01 M sodium hydroxide is required to change the colour Inductively coupled plasma-atomic emission spectrometry
of the indicator to blue. To 100 mL of solution SI add (2.2.57).
0.2 roL of methyl orange solution R. Not more than 1.0 mL of Test solution Use solution S3.
0.01 M hydrochloric acid is required to reach the beginning of
the colour change of the indicator from yellow to orange.
vanadium standard solution (l giL V) R, diluting with a
Absorbance (2.2.25) Maximum 0.2, determined between mixture of 2 volumes of hydrochloric acid R and 8 volumes of
wavelengths of 220 nm and 340 nm on solution SI. water R .
Reducing substances To 20 mL of solution SI add 1 mL Wavelength Use the emission of vanadium at 292.40 nm,
of dilute sulfuric acid R and 20 mL of 0.002 M potassium the spectral background being taken as 292.35 nm.
permanganate. Boil under a reftux condenser for 3 min and
Verify the absence of vanadium in the hydrochIoric acid
cool immediately. Add 1 g of potassium iodide R and titrate
used.
starch solution R as indicator. Carry out a blank titration. Extractable zinc Maximum 1 ppm.
The difference between the titration volumes is not more Atomic absorption spectrometry (2.2.23, Method 1).
than 0.5 mL. Test solution Use solution S3.
Substances soluble in hexane Place 10 g in a 250 mL Reference solutions Prepare the reference solutions using
conical borosilicate-glass ftask with a ground-glass neck. zinc standard solution (lO ppm Zn) R, diluting with 0.1 M
Add 100 mL of hexane R and boil under a reftux condenser hydrochloric acid.
for 4 h, stirring constant1y. Cool in iced water and filter Source Zinc hollow-cathode lamp.
rapidly through a sintered-glass filter (16) (2.1.2)
maintaining the solution at O oC (the filtration time must be
les s than 5 min; if necessary the filtration may be accelerated Atomisation device Air-acetylene ftame .
by applying pressure to the solution). Evaporate 20 mL of Extractable zirconium Maximum 0.1 ppm.
the filtra te in a tared borosilicate-glass dish on a water-bath. Inductively coupled plasma-atomic emission spectrometry
Dry the residue in an oven at 100-105 oC for 1 h. The mass (2.2.57).
V-A564 Appendix xx e 2014
Test solution Use solution S3. Referenee solution (h) Dissolve 60.0 mg of plastie
Referenee solutions Prepare the reference solutions using additive 10 CRS in 10.0 mL of the solvent mixture. Dilute
zireonium standard solution (1 giL ZI:) R, diluting with a 2.0 mL of this solution to 50.0 mL with the solvent mixture.
mixture of 2 volumes of hydroehlorie aeid R and 8 volumes of Referenee solution (i) Dissolve 60.0 mg of plastie additive
water R. 11 CRS in 10.0 mL of methylene ehloride R. Dilute 2.0 mL of
Wavelength Use the emission of zirconium at 343.82 nm, this solution to 50.0 mL with methylene ehloride R .
the spectral background being taken as 343.92 nm. Referenee solution W Dissolve 60.0 mg of plastie additive
Verify the absence of zirconium in the hydrochloric acid 12 CRS in 10.0 mL of methylene ehloride R. Dilute 2.0 mL of
used. this solution to 50.0 mL with methylene ehloride R.
Extractable heavy metals (2.4.8) Maximum 2.5 ppm. A.
If the substance to be examined contains plastic additive
Evaporate 50 mL of solution S3 to about 5 mL on a water- 07 and/or plastic additive 08, proceed as follows.
bath and dilute to 20.0 mL with water R. 12 mL of the Column:
solution complies with test A. Prepare the reference solution - size: 1 = 0.25 m, 0 = 4.6 mm;
using 2.5 mL of lead standard solution (10 ppm Pb) R. - stationary phase: oetadeeylsilyl silica gel for chromatography R
Sulfated ash (2.4.14) Maximum 1.0 per cent, determined (5 J..lm).
on 5.0 g. Mobile phase water R, acetonitrile R (30:70 VIV).
This Iimit does not apply to material opacified with titanium Flow rate 2 mLlmin.
dioxide. Detection Spectrophotometer at 280 nm.
Supplementary tests Injeetion 20 J..lL of test solution S21, ofthe corresponding
blank solution, of reference solution (a), and either reference
These tests are to be earried out, in whole or in part, only if
solution (d) or (e), or reference solutions (d) and (e) .
required by the stated eomposition of the material.
Run time 30 mino
Phenolic antioxidants Liquid chromatography (2.2.29).
System suitability:
Solvent mixture aeetonitrile R, tetrahydrofuran R (50:50
VIV) . - resolution: minimum 8.0 between the peaks due to plastic
additive 07 and plastic additive 08 in the chromatogram
obtained with reference solution (a);
dryness in vaeuo at 45 oc. Dissolve the residue with 5.0 mL
of the solvent mixture. Prepare a blank solution from the - the chromatogram corresponding to test solution S21 only
blank solution corresponding to solution S2. show peaks due to antioxidants stated in the composition
and minor peaks that also appear in the chromatogram
corresponding to the blank solution.
dryness in vaeuo at 45 oc. Dissolve the residue with 5.0 mL
of methylene ehloride R. Prepare a blank solution from the Limit The are as of the peaks of test solution S21 are less
blank solution corresponding to solution S2. than the areas of the corresponding peaks in the
chromatograms obtained with reference solutions (d) and/or
Of the following referenee solutions, only prepare those that are
(e) .
neeessary for the analysis of the phenolie antioxidants stated in the
eomposition of the substanee to be examined. B. If the substance to be examined contains one or more of
the following antioxidants:
Referenee solution (a). Dissolve 25 .0 mg of
butylhydroxytoluene CRS (plastic additive 07) and 60.0 mg of - plastic additive 09;
plastie additive 08 CRS in 10.0 mL ofthe solvent mixture. - plastic additive 10;
Dilute 2.0 mL of this solution to 50.0 mL with the solvent - plastic additive 11;
mixture. - plastic additive 12;
Referenee solution (b) Dissolve 60.0 mg of plastie additive - plastic additive 13;
09 CRS and 60.0 mg of plastie additive 10 CRS in 10.0 mL
proceed as described aboye with the following modifications.
of the solvent mixture. Dilute 2.0 mL of this solution to
50.0 mL with the solvent mixture. Mobile phase water R, tetrahydrofuran R, acetonitrile R
(10:30:60 VIVI V) .
Referenee solution (e) Dissolve 60.0 mg of plastie additive
I1 CRS and 60.0 mg of plastie additive 12 CRS in 10.0 mL Flow rate 1.5 mUmin.
of methyleneehloride R. Dilute 2.0 mL of this solution to Injeetion 20 J..lL of test solution S21, of the corresponding
50.0 mL with methylene ehloride R. blank solution, of reference solution (b) and reference
Referenee solution (d) Dissolve 25 .0 mg of solutions of the antioxidants on the Iist aboye that are stated
butylhydroxytoluene CRS (plastic additive 07) in 10.0 mL of in the composition.
the solvent mixture . Dilute 2.0 mL of this solution to System suitability:
50.0 mL with the solvent mixture. - resolution: minimum 2.0 between the peaks due to plastic
Referenee solution (e) Dissolve 60.0 mg of plastie additive additive 09 and plastic additive 10 in the chromatogram
08 CRS in 10.0 mL of the solvent mixture. Dilute 2.0 mL of obtained with reference solution (b);
this solution to 50.0 mL with the solvent mixture. - the chromatogram corresponding to test solution S21 only
Referenee solution (j) Dissolve 60.0 mg of plastie additive show peaks due to antioxidants stated in the composition
13 CRS in 10.0 mL of the solvent mixture. Dilute 2.0 mL of and minor peaks that also appear in the chromatogram
this solution to 50.0 mL with the solvent mixture. corresponding to the blank solution.
Referenee solution (g) Dissolve 60.0 mg of plastie additive
09 CRS in 10.0 mL ofthe solvent mixture. Dilute 2.0 mL of
this solution to 50.0 mL with the solvent mixture.
Limit The areas of the peaks of test solution S21 are less Drying A In airo
than the areas of the corresponding peaks in the Development B Over a path of 17 cm with mobile
chromatograms obtained with reference solutions of the phase B.
antioxidants on the list aboye that are stated in the Drying B In airo
composition.
Detection Examine in ultraviolet light at 254 nm, spray
C. If the substance to be examined contains plastic additive with a/coholic iodine solution R and examine in ultraviolet light
11 and/or plastic additive 12, carry out the test as at 254 nm after 10-15 mino
described for plastic additive 07 andlor plastic additive
System suitability Reference solution (o):
08 with the foHowing modifications.
- the chromatogram shows 2 clearly separated spots.
Mobile phase water R, 2-propanol R, methanol R (5:45 :50
VIV/V). Limits Any spots in the chromatogram obtained with test
solution S23 are not more intense than the spots in the same
locations in the chromatograms obtained with the reference
Injection 20 ~lL of test solution S22, of the corresponding solutions.
blank solution, of reference solution (e), and either of
reference solution (i) or (j), or reference solutions (i) and (j).
Amides and stearates Thin-Iayer chroma1Ography
(2.2.27) .
System suitability:
Test solution Use test solution S23 described in the test
- resolution: minimum 2.0 between the peaks due to plastic for non-phenolic antioxidants.
additive 11 and plastic additive 12 in the chroma1Ogram
Reference solution (p) Dissolve 20 mg of stearie acid CRS
obtained with reference solution (e);
(plastic additive 19) in methylene ehloride R and dilute to
- the chroma1Ogram corresponding to test solution S22 only 10 mL with the same solvent.
show peaks due 10 antioxidants stated in the composition
Reference solution (q) Dissolve 40 mg of plastic additive
and minor peaks that also appear in the chromatogram
20 CRS in methylene ehloride R and dilute to 20 mL with the
corresponding to the blank solution.
same solvent.
Limit The areas of the peaks of test solution S22 are less
than the areas of the corresponding peaks in the
Reference solution (r) Dissolve 40 mg of plastie additive
21 CRS in methylene chloride R and dilute to 20 mL with the
chroma1Ograms obtained with reference solutions (i) andlor
same solvent.
0) .
Plates TLC siliea gel GF254 plates R (2 plates).
Non-phenolic antioxidants Thin-Iayer chroma1Ography
(2.2.27) . A. Mobile phase anhydrous ethanol R, trimethylpentane R
Test solution S23 Evaporate 100 mL of solution S2 to (25:75 VN).
dryness in vacuo at 45 oC. Dissolve the residue in 2 mL of Application 1O ~lL of test solution S23 and reference
aeidified methylene ehloride R. solution (p).
Reference solution (k) Dissolve 60 mg of plastic additive Development Over a path of 10 cm.
14 CRS in methylene ehloride R and dilute 10 10 mL with the Drying In airo
same solvent. Dilute 2 mL of this solution to 10 mL with Detection Spray with a 2 gIL solution of
acidified methylene ehloride R. diehlorophenolindophenol sodium salt R in anhydrous ethanol R
Reference solution (1) Dissolve 60 mg of plastie additive and heat in an oven at 120 cc for a few minutes 10 intensify
15 CRS in methylene ehloride R and dilute to 10 mL with the the spots.
same solvent. Dilute 2 mL of this solution to 10 mL with Limit Any spot corresponding to plastic additive 19 in the
acidified methylene ehloride R. chroma1Ogram obtained with test solution S23 is identical in
Reference solution (m) Dissolve 60 mg of plastie additive position (R p = about 0.5) but not more intense than the
16 CRS in methylene ehloride R and dilute to 10 mL with the spot in the same location in the chromatogram obtained with
same solvent. Dilute 2 mL of this solution to 10 mL with reference solution (p) .
acidified methylene ehloride R. B. Mobile phase A hexane R.
Reference solution (n) Pissolve 60 mg of plastic additive Mobile phase B methanol R, methylene ehloride R (5 :95
17 CRS in methylene ehloride R and dilute 10 10 mL with the V/V).
same solvent. Dilute 2 mL of this solution to 10 mL with
acidified methylene ehloride R. Application 10 ¡.¡L of test solution S23 and reference
solutions (q) and (r).
Reference solution (o) Dissolve 60 mg of plastie additive
Development A Over a path of 13 cm with mobile phase
16 CRS and 60 mg of plastic additive 17 CRS in methylene
ehlonde R and dilute to 10 mL with the same solvent. Dilute A.
2 mL of this solution to 10 mL with acidified methylene Drying A In airo
chloride R . Development B Over a path of 10 cm with mobile
Plate TLC siliea gel GF254 plate R. phase B.
Mobile phase A hexane R. Drying B In air.
Mobile phase B methylene chloride R . Detection Spray with a 40 giL solution of phosphomolybdic
aeid R in anhydrous ethanol R and heat in an oven at 120 oC
Application 20 ¡.¡L of test solution S23, of reference
until spots appear.
solution (o) and of the reference solutions corresponding to
aH the phenolic and non-phenolic antioxidants mentioned in Limit Any spots corresponding 10 plastic additive 20 or
the type composition of the material to be examined. plastic additive 21 in the chromatogram obtained with the
test solution S23 are identical in position (R p = about 0.2)
Development A Over a path of 18 cm with mobile
but not more intense than the corresponding spots in the
phase A.
chromatograms obtained with reference solutions (q) and (r).
V-A566 Appendix xx D 2014

D. Polypropylene for Containers and - tale: maximum 0.5 per cent;
Closures for Parenteral Preparations and - magnesium oxide: maximum 0.2 per cent;
Ophthalmic Preparations - calcium stearate or zinc stearate or a mixture of both:
maxirnum 0.5 per cent;
- titanium dioxide, only for material s for containers for
Definition ophthalmic use: maximum 4 per cent.
Polypropylene consists of the homopolyrner of propylene or The supplier of the material must be able to demonstrate
of a copolyrner of propylene with not more than 25 per cent that the qualitative and quantitative composition of the type
of ethylene or of a mixture (alloy) of polypropylene with not sample is satisfactory for each production batch.
more than 25 per cent of polyethylene. It may contain Characters
additives.
Appearance Powder, beads, granules or, after
Production transformation, translucent sheets of varying thicknesses or
A cenain number of additives are added ro the polyrner in containers.
order to optimise their chemical, physical and mechanical Solubility Practically insoluble in water, soluble in hot
propenies in order ro adapt them for the intended use. aromatic hydrocarbons, practically insoluble in anhydrous
All these additives are chosen from the appended list which ethanol, in hexane and in methano!.
specifies for each product the maximum allowable contento It softens at temperatures beginning at about 120 oC.
They may contain at most 3 antioxidants, one or several
I dentification
lubricants or antiblocking agents as well as titanium dioxide
as opacifYing agent when the material must provide Jf necessary, cut the material to be examined into pieces of
protection from light. maximum dimension on a side of not greater than 1 cm.
- butylhydroxytoluene (plastic additive 07): maximum A. Infrared absorption spectrophotometry (2.2.24).

0.125 per cent; Preparation To 0.25 g add 10 mL of toluene R and boil
- pentaerythrityl tetrakis [3-(3,5-di-tert-butyl-4- under a refiux condenser for about 15 min. Place a few
hydroxyphenyl)propionate] (pI as tic additive 09): drops of the hot solution on a sodium chloride disc and
maximum 0.3 per cent; evaporate the solvent in an oven at 80 ce.
- 1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-s-triazine- Absorption maxima At 1375 cm- J , 1170 cm- l ,
2,4,6(1H,3H,5H)-trione (plastic additive 13): maximum 995 cm- J and 970 cm-l .
0.3 per cent; The spectrum obtained is identical to the spectrum obtained
- octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate, with the material selected for the type sample. If the material
(plastic additive 11): maximum 0.3 per cent; to be examined is in the form of sheets, the identification
may be performed directly on a cut piece of suitable size.
- ethylene bis [3,3-bis [3-( 1, l-dimethylethyl)-4-
hydroxyphenyl]butanoate] (plastic additive 08): maximum B. It complies with the supplementary tests corresponding to
0.3 per cent; the additives present (see Tests) .
- dioctadecyl disulfide (plastic additive 15) : maximum e. In a platinum crucible, mix about 20 mg with 1 g of
0.3 per cent; potassium hydrogen sulfate R and heat until completely
melted. Allow to cool and add 20 mL of dilute sulfuric
- 2,2',2",6,6' ,6 "-hexa-tert-butyl-4,4' ,4"- [(2,4,6-trimethyl-
acid R. Heat gently. Filter the resulting solution. To the
1,3 ,5-benzenetriyl)trismethylene] triphenol (pI as tic additive
filtrate add 1 mL of phosphoric acid R and 1 mL of strong
10): maximum 0.3 per cent;
hydrogen peroxide solution R . If the substance is opacified
- 2,2'-bis(octadecyloxy)-5,5 '-spirobi[I,3,2- with titanium dioxide, an orange-yellow colour develops.
dioxaphosphinane] (plastic additive 14): maxirnum
0.3 per cent; Tests
- didodecyl 3,3'-thiodipropionate (plastic additive 16): Jf necessaly, cut the material to be examined into pieces of
maximum 0.3 per cent; maximum dimensiol1 011 a side of not greater than 1 cm.
- dioctadecyl 3,3'-thiodipropionate (plastic additive 17): Solution S1 Use solutiol1 SI within 4 h of preparation. Place
maxirnum 0.3 per cent; 25 g in a borosilicate-glass fiask with a ground-glass neck.
Add 500 mL of water for injections R and boil under a refiux
- tris(2,4-di-tert-butylphenyl) phosphite (plastic additive 12):
condenser for 5 h. AlIow ro cool and decant. Reserve a
The total of antioxidant additives listed aboye does not and filter the rest through a sintered-glass filter (16) (2.1.2).
exceed 0.3 per cent.
Solution S2 Place 2.0 g in a conical borosilicate-glass fiask
- hydrotaleite: maximum 0.5 per cent; with a ground-glass neck. Add 80 mL of toluene R and boil
- alkanamides: maximum 0.5 per cent; under a refiux condenser with constant stirring for 1 h
- alkenamides: maximum 0.5 per cent; 30 mino AlIow to cool to 60 oC and add with continued
- sodium silico-aluminate: maximum 0.5 per cent; stirring 120 mL of methanol R. Filter the solution through a
sintered-glass filter (16) (2.1.2) . Rinse the fiask and the filter
- silica: maximum 0.5 per cent;
with 25 roL of a mixture of 40 mL of toluene R and 60 mL
- sodium benzoate: maximum 0.5 per cent; of methanol R, add the rinsings to the filtrate and dilute ro
- fatty acid esters or salts: maximum 0.5 per cent; 250 .0 mL with the same mixture of solvents. Prepare a blank
- trisodium phosphate: maximum 0.5 per cent; solution.
- liquid paraffin: maximum 0.5 per cent;
2014 Appendix XX D V-A567
Solution S3 Place 100 g in a conical borosilicate-glass Test solution Use solution S3.
flask with a ground-glass neck. Add 250 mL of 0.1 M Reference solutions Prepare the reference solutions using
hydrochloric acid and boil under a reflux condenser with titanium standard solution (100 ppm Tz) R , diluting with
constant stirring for 1 h. Allow to cool and decant the 0.1 M hydrochloric acid.
solution. Wavelength Use the emission of titanium at 336.12 nm,
Appearance of solution Solution SI is not more the spectral background being taken as 336.16 nm.
opalescent than reference suspension II (2.2.1) and is Verify the absence of titanium in the hydrochloric acid used.
colourless (2. 2.2, M ethod 11).
Extractab1e vanadium Maximum 0.1 ppm.
Acidity or aIkalinity To 100 mL of solution SI add
0.15 mL of BRP indicator solution R. Not more than 1.5 mL Inductively coupled plasma-atomic emission spectrometry
(2.2.57) .
of 0.01 M sodium hydroxide is required to change the colour
ofthe indicatorto blue . To 100 mL of solution SI add Test solution Use solution S3.
0.2 mL of methyl orange solution R. Not more than 1.0 mL of Reference solutions Prepare the reference solutions using
0.01 M hydrochloric acid is required to reach the beginning of vanadium standard solution (1 giL V? R, diluting with a
the colour change of the indicator from yellow to orange. mixture of 2 volumes of hydrochloric acid R and 8 volumes of
Absorbance (2.2.25) Maximum 0.2, determined water R.
between wavelengths of 220 nm to 340 nm on solution SI. Wavelength Use the emission of vanadium at 292.40 nm,
Reducing substances To 20 mL of solution SI add 1 mL the spectral background being taken as 292.35 nm.
of dilute sulfuric acid R and 20 mL of 0.002 M potassium Verify the absence of vanadium in the hydrochloric acid
perrnanganate. Boil under a refiux condenser for 3 min and used.
coo] immediate]y. Add 1 g of potassium iodide R and titrate Extractable zinc Maximum 1 ppm.
immediately with 0.01 M sodium thiosulfate, using 0.25 mL of Atomic absorption spectrometry (2.2.23, Method 1) .
The difference between the titration volumes is not more Test solution Use solution S3.
than 0.5 mL. Reference solutions Prepare the reference solutions using
zinc standard solution (10 ppm Zn) R, diluting with 0.1 M
Substances soluble in hexane Place 10 g in a 250 mL
hydrochloric acid.
conical borosilicate-glass flask with a ground-glass neck.
Add 100 mL of hexane R and boil under a reflux condenser Source Zinc hollow-cathode lampo
for 4 h, stirring constant1y. Cool in iced water and filter Wavelength 213.9 nm.
rapidly through a sintered-glass filter (16) (2.1.2) maintaining Atomisation device Air-acetylene flameo
the solution at O oC (the filtration time must be less than
Verify the absence of zinc in the hydrochloric acid used.
5 min; if necessary the filtration may be accelerated by
applying pressure to the solution). Evaporate 20 mL of the Extractable heavy metals (2.4.8) Maximum 2.5 ppm.
filtrate in a tared glass dish on a water-bath. Dry the residue Concentrate 50 mL of solution S3 to about 5 mL on a
in an oven at 100-105 oC for 1 h. The mass of the residue water-bath and dilute to 20.0 mL with water R. 12 mL ofthe
obtained must be within 10 per cent ofthe residue obtained solution complies with test A. Prepare the reference solution
with the type sample and do es not exceed 5 per cent. using 2.5 mL of lead standard solution (10 ppm Pb) R.
Extractable aluminium Maximum 1 ppm. Sulfated ash (2.4.14) Maximum 1.0 per cent, determined
Inductively coupled plasma-atomic emission spectrometry on 5.0 g. This limit does not apply to material that has been
(2.2.57). opacified with titanium dioxide.
Test solution Use solútion S3 . Supplementary Tests
Reference solutions Prepare the reference solutions using These tests are to be camed out, in whole or in part, only ij
aluminium standard solution (200 ppm Al) R, diluting with required by the stated composition of the material.
O. 1 M hydrochloric acid. Phenolic antioxidants Liquid chromatography (2.2.29).
Wavelength Use the emission of aluminium at 396.15 nm, Solvent mixture acetonitrile R, tetrahydrofuran R (50:50
the spectral background being taken as 396.25 nm. VIV).
Verify the absence of aluminium in the hydrochloric acid Test solution S21 Evaporate 50 mL of solution S2 to
used. dryness in vacuo at 45 oc. Dissolve the residue with 5.0 mL
Extractable chromium Maximum 0.05 ppm. of the solvent mixture. Prepare a blank solution from the
Inductively coupled plasma-atomic emission spectrometry blank solution corresponding to solution S2.
(2.2.57) . Test solution S22 Evaporate 50 mL of solution S2 to
Test solution Use solution S3 . dryness in vacuo at 45 oc. Dissolve the residue with 5.0 mL
of methylene chloride R . Prepare a blank solution from the
Reference solutions Prepare the reference solutions using blank solution corresponding to solution S2 .
chromium standard solution (100 ppm Cr) R, diluting with a
mixture of 2 volumes of hydrochloric acid R and 8 volumes of Of the following reference solutions, only prepare those that
water R .
are necessary for the analysis of the phenolic antioxidants
stated in the composition of the substance to be examÍned.
Wavelength Use the emission of chromium at 205.55 nm,
the spectral background being taken as 205.50 nm. Reference solution (a) Dissolve 25 .0 mg of
butylhydroxytoluene CRS (plastic additive 07) and 60.0 mg of
Verify the absence of chromium in the hydrochloric acid plastic additive 08 CRS in 10.0 mL of the solvent mixture.
used. Dilute 2.0 mL of this solution to 50.0 mL with the solvent
Extractab1e titanium Maximum 1 ppm. mixture.
(2.2.57) .
V-A568 Appendix xx D 2014
Referenee solution (b) Dissolve 60.0 mg of plastic additive - plastie additive 12;
09 CRS and 60.0 mg of plastic additive 10 CRS in 10.0 mL - plastie additive 13;
of the solvent mixture. Dilute 2.0 mL of this solution to carry out the test as deseribed aboye with the following
50.0 mL with the solvent mixture.
modifieations.
Referenee solution (e) Dissolve 60.0 mg of plastic additive Mobile phase water R, tetrahydrofuran R, acetonitrile R
11 CRS and 60.0 mg of plastic additive 12 CRS in 10 mL of (10 :30:60 VIV/V) .
methylene chloride R. Dilute 2.0 mL of this solution 10
50.0 mL with methylene chloride R. Flow rate 1.5 mUmin.
Referenee solution (d) Dissolve 25.0 mg of Injeetion 20 /lL of test solution S21, eorresponding blank
butylhydroxytoluene CRS (plastie additive 07) in 10.0 mL of solution, referenee solution (b) and referenee solutions of the
the solvent mixture. Dilute 2.0 mL of this solution to antioxidants on the list aboye that are stated in the
50.0 mL with the solvent mixture. eomposition.
Referenee solution (e) Dissolve 60.0 mg of plastic additive System suitability:
08 CRS in 10.0 mL of the solvent mixture. Dilute 2.0 mL of - resolution: minimum 2.0 between the peaks due to plastie
this solution to 50.0 mL with the solvent mixture. additive 09 and plastic additive 10 in the ehromatogram
Referenee solution (f) Dissolve 60.0 mg of plastic additive obtained with referenee solution (b);
13 CRS in 10.0 mL ofthe solvent mixture. Dilute 2.0 mL of - the ehromatogram eorresponding 10 test solution S21 only
this solution to 50.0 mL with the solvent mixture. show peaks due 10 antioxidants stated in the eomposition
Referenee solution (g) Dissolve 60.0 mg of plastic additive and minor peaks that also appear in the ehroma1Ogram
09 CRS in 10.0 mL of the solvent mixture. Dilute 2.0 mL of eorresponding 10 the blank solution.
this solution to 50.0 mLwith the solvent mixture. Limit The areas of the peaks in the ehromatogram
Referenee solution (h) Dissolve 60.0 mg of plastic obtained with test solution S21 are les s than the areas of the
additive 10 CRS in 10.0 mL of the solvent mixture. Dilute eorresponding peaks in the ehroma1Ograms obtained with
2.0 mL ofthis solution to 50.0 mL with thesolvent mixture. referenee solutions of the antioxidants on the list aboye that
are stated in the eomposition.
Referenee solution (i) Dissolve 60.0 mg of plastic additive
11 CRS in 10.0 mL of methylene chloride R. Dilute 2.0 mL of C. If the substanee to be examined eontains plastie additive
this solution to 50.0 mL with methylene chloride R. 11 and/or plastie additive 12, carry out the test as
deseribed for plastie additive 07 and/or plastie additive
Referenee solution (j) Dissolve 60.0 mg of plastic additive
08 with the following modifieations.
12 CRS in 10.0 mL of methylene chloride R. Dilute 2.0 mL of
this solution to 50.0 mL with methylene chloride R. Mobile phase water R, 2-propanol R, methanol R (5:45:50
VIV/V) .
A. If the substanee 10 be examined eontains plastie additive
07 andlor plastie additive 08, carry out the test as Flow rate 1.5 mUmin.
follows . Injeetion 20 /lL of test solution S22, eorresponding blank
Column: solution, referenee solution (e), and either referenee solution
(i) or 0), or referenee solutions (i) and O).
- size: 1 = 0.25 m, 0 = 4.6 mm;
System suitability:
- stationary phase: octadecylsilyl silica gel for chromatography R
(5 /lm) . - resolution: minimum 2.0 between the peaks due 10 plastie
additive 11 and plastic additive 12 in the ehroma1Ogram
Mobile phase water R, acetonitrile R (30:70 V/V).
obtained with referenee solution (e);
Flow rate 2 mUmin.
- the ehroma1Ogram eorresponding to test solution S22 on1y
Deteetion Speetrophotometer at 280 nm. show peaks due to antioxidants stated in the eomposition
Injeetion 20 /lL of test solution S21, eorresponding blank and minor peaks that also appear in the ehromatogram
solution and referenee solution (a), and either referenee eorresponding 10 the blank solution.
solution (d) or (e), or referenee solutions (d) and (e) . Limit The areas of the peaks in the ehroma1Ogram
Run time 30 min. obtained with test solution S22 are less than the areas of the
System suitability: eorresponding peaks in the ehromatograms obtained with
- resolution: minimum 8.0 between the peaks due 10 plastic referenee solutions (i) and/or O) .
additive 07 and plastie additive 08 in the ehroma1Ogram Non-phenolic antioxidants Thin-layer ehroma1Ography
obtained with referenee solution (a); (2.2.27).
- the ehroma1Ogram eorresponding 10 test solution S21 on1y Test solution S23 Evaporate 100 mL of solution S2 10
show peaks due to antioxidants stated in the eomposition dryness in vacuo at 45 oC. Dissolve the residue with 2 mL of
and minor peaks that also appear in the ehromatogram acidified methylene chloride R.
eorresponding to the blank solution. Referenee solution (k) Dissolve 60 mg of plastic additive
Limit The areas of the peaks in the ehroma1Ogram 14 CRS in methylene chloride R and dilute to 10 mL with the
obtained with test solution S21 are les s than the areas of the same solvent. Dilute 2 roL of the solution to 10 mL with
eorresponding peaks in the ehromatograms obtained with acidified methylene chloride R.
referenee solutions (d) andlor (e). Referenee solution (l) Dissolve 60 mg of plastic additive
B. If the substanee to be examined eontains one or more of 15 CRS in methylene chloride R and dilute 10 10 mL with the
the following antioxidants: same solvento Dilute 2 mL of the solution to 10 mL with
- plastie additive 09; acidified methylene chloride R.
- plastie additive 10;
- plastie additive 11;
2014 Appendix XX E V-A569
Reference solution (m) Dissolve 60 mg of plastie additive position (Rp about 0.5) but not more intense than the spot
16 CRS in methylene ehlonde R and dilute to 10 mL with the in the same position in the chromatogram obtained with
same solvent. Dilute 2 mL of the solution to 10 mL with reference solution (p).
aeidified methylene ehlonde R. B. Mobile phase A hexane R.
Reference solution (n) Dissolve 60 mg of plastie additive Mobile phase B methanol R, methylene ehloride R (5:95
17 CRS in methylene ehlonde R and dilute to 10 mL with the VIV).
same solvent. Dilute 2 mL of the solution to 10 mL with
Application 10!1L of solution S23 and reference solutions
acidified methylene ehlonde R. (q) and (r).
Reference solution (o) Dissolve 60 mg of plastie additive
16 CRS and 60 mg of plastie additive 17 CRS in methylene
A.
ehlonde R and dilute to 10 mL with the same solvent. Dilute
2 mL of the solution to 10 mL with aeidified methylene Drying A In airo
ehlonde R . Development B Over a path of 10 cm with mobile
Plate TLC siliea gel GF254 plate R. phase B.
Mobile phase A hexane R. Drying B In air.
Mobile phase B methylene ehlonde R . Detection Spray with a 40 giL solution of phosphomo1ybdie
acid R in anhydrous ethanol R; heat in an oven at 120 oC
Application 20!1L of test solution S23, reference solution
until spots appear.
(o) and reference solutions corresponding to aH the phenolic
and non-phenolic antioxidants mentioned in the type Limit Any spots corresponding to plastic additive 20 or
composition of the material to be examined. plastic additive 21 in the chromatogram obtained with test
solution S23 are identical in position (R p about 0.2) but not
more intense than the corresponding spots in the
A.
chromatograms obtained with reference solutions (q) and (r) .
Drying A In airo
Development B Over a path of 17 cm with mobile
phase B.
Drying B In airo
E. Poly(ethylene . vinyl acetate) for
Detection Examine in ultraviolet light at 254 nm; spray Containers and Tubing for Total
with aleoholie iodine solution R and examine in ultraviolet light Parenteral Nutrition Preparations
at 254 nm after 10-15 mino
System suitability Reference solution (o): (Ph. Eur. method 3.1.7)
- the chromatogram shows 2 cIearIy separated spots . Definition
Limits Any spots in the chromatogram obtained with test Poly(ethylene - vinyl acetate), complying with the foIlowing
solution S23 are not more intense than the spots in the same requirements, is suitable for the manufacture of containers
positions in the chromatograms obtained with the reference and tubing for total parenteral nutrition preparations. It is
solutions. obtained by copolyrnerisation of mixtures of ethylene and
Amides and stearates Thin-Iayer chromatography vinyl acetate.
(2.2.27). Content of vinyl acetate:
Test solution Use solution S23 described in the test for - material used for containers: a defined quantity of not
non-phenolic antioxidants. more than 25 per cent;
Reference solution (p) Dissolve 20 mg of stearie acid CRS - material used for tubing: a defined quantity of not more
(plastic additive 19) in methylene ehlonde R and dilute to than 30 per cent.
10 mL with the same solvent.
Reference solution (q) Dissolve 40 mg of plastie additive Production
20 CRS in methylene ehlonde R and dilute to 20 mL with the A certain number of additives are added to the polyrner in
same solvent. order to optimise their chemical, physical and mechanical
Reference solution (r) Dissolve 40 mg of plastie additive properties in order to adapt them for the intended use.
21 CRS in methylene ehlonde R and dilute to 20 mL with the AH these additives are chosen from the appended list which
same solvent. specifies for each product the maximum aHowable contento
Plate TLC siliea gel GF254 plate R (2 plates). Poly(ethylene - vinyl acetate) may contain not more than 3 of
A. Mobile phase anhydrous ethanol R, tnmethylpentane R the foIlowing antioxidants:
(25:75 VIV) . - butylhydroxytoluene (plastic additive 07): maximum
Application 10!1L of solution S23 and reference 0.125 per cent;
solution (p) . - pentaerythrityl tetrakis[3-(3,5-di-tert-butyl-4-
Development Over a path of 10 cm. hydroxyphenyl)propionate] (plastic additive 09):
Drying In airo
- octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate
(plastic additive 11): maximum 0.2 per cent;
dichlorophenolindophenol sodium salt R in anhydrous ethanol R
and heat in an oven at 120 oC for a few minutes to intensify - tris(2,4-di-tert-butylphenyl) phosphite (plastic additive 12):
the spots . maximum 0.2 per cent;
Limit Any spot corresponding to plastic additive 19 in the _ 2,2 f ,2" ,6,6 f ,6" -hexa-tert-butyl-4,4 f ,4" - [(2,4,6-trimethyl-
chromatogram obtained with test solution S23 is identical in 1,3,5-benzenetriyl)trismethylene]triphenol (plastic additive
10) : maximum 0.2 per cent.
V-A570 Appendix xx E 2014
Itmay also contain: Appearance of solution S2 Solution S2 is c1ear (2.2.1)

- oleamide (plastic additive 20) : maximum 0.5 per cent; and colourless (2.2.2, Method JI) .
- erucamide (plastic additive 21): maximum 0.5 per cent; Acidity or aIkalinity To 100 mL of solution S2 add
0.15 mL of BRP indicator solution R . Not more than 1.0 mL
- calcium stearate or zinc stearate or a mixture of both:
of 0.01 M sodium hydroxide is required 10 change the colour
of the indicator to blue. To 100 mL of solution S2 add
- calcium carbonate or potassium hydroxide: maximum 0.2 mL of methyl orange solution R . Not more than 1.5 mL of
0.5 per cent; 0.01 M hydrochloric acid is required to reach the beginning of
- colloidal silica: maximum 0.2 per cent. the colour change of the indicator from yellow to orange.
The supplier of the material must be able 10 demonstrate Absorbance (2.2.25) Maximum 0.2 determined between
that the qualitative and quantitative composition of the type wavelengths of 220 nm and 340 nm on solution S2.
sample is satisfactory for each production batch. Reducing substances To 20 mL of solution S2 add 1 mL
Characters of d¡lute sulfuric acid R and 20 mL of 0.002 M potassium
permanganate. Boil under a refiux condenser for 3 min and
Appearance Beads, granules or, after transformation,
cool immediately. Add 1 g of potassium iodide R and titrate
translucent sheets or tubing of varying thickness or samples
of finished ob;ects.
SolubiZity Practically insoluble in water, soluble in hot The difference between the titration volumes is not more
aromatic hydrocarbons, practically insoluble in anhydrous than 0.5 mL.
ethanol, in methanol and in hexane, which dissolves,
Anúdes and stearic acid Thin-Iayer chromatography
however, low molecular mas s polymers.
(2.2.27).
It burns with a blue fiame.
Test solution Evaporate 100 mL of solution Sito dryness
The temperature at which the substance softens changes with in vacuo at 45 oC. Dissolve the residue in 2 mL of acidified
the vinyl acetate content: from about 100 oC for contents of methylene chloride R.
a few per cent 10 about 70 oC for contents of 30 per cent.
Reference solution (a) Dissolve 20 mg of stearic acid CRS
Identification (plastic additive 19) in 10 mL of methylene chloride R .
Jf necessary, cut the samples of material to be examined into pieces Reference solution (b) Dissolve 40 mg of plastic additive
of maximum dimension on a side of not greater than 1 cm. . 20 CRS in 10 mL of methylene chlO1'Íde R. Dilute 1 mL of
Infrared absorption spectrophotometry (2.2.24). this solution 10 5 mL with methylene chloride R.
Preparation To 0.25 g add 10 mL of toluene R and boil Reference solution (c) Dissolve 40 mg of plastic additive
21 CRS in 10 mL of methylene chlO1'Íde R. Dilute 1 mL of
under a refiux condenser for about 15 mino Place a few
drops of the solution obtained on a disc of sodium chloride this solution to 5 mL with methylene chloride R.
and evaporate the solvent in an oven at 80 oc. Plates TLC silica gel GF254 plate R (2 plates).
Absorption maxima due to vinyl acetate At A. Mobile phase anhydrous ethanol R, trimethylpentane R
1740 cm- 1, 1375 cm-I, 1240 cm- I, 1020 cm- I and (25:75 V/V).
610 cm-l. Application 10 ¡lL.
Absorption maxima due to ethylene At Development Over a path of 10 cm.
2920-2850 cm- 1, 1470 cm- I, 1460 cm- I , 1375 cm- t, Drying In air.
730 cm- 1 and 720 cm- l.
The spectrum obtained is identical to the spectrum obtained dichlorophenolindophenol sodium salt R in anhydrous
with the type sample provided by the manufacturer. If the ethanol R and heat in an oven at 120 oC for a few
material to be examined is in the form of sheets, the minutes to intensifY the spots.
spectrum may be determined directly on a cut piece of Limit Any spot corresponding 10 plastic additive 19 in
suitable size. the chromatogram obtained with the test solution is not
Tests more intense than the spot in the chromatogram
obtained with reference solution (a).
Ij necessary, cut the samples of the material to be examined into
pieces of maximum dimension on a side of not greater than 1 cm. B. Mobile phase A hexane R .
Solution SI Place 2.0 g in a borosilicate-glass fiask with a Mobile phase B methanol R, methylene chloride R (5:95
ground-glass neck. Add 80 mL of toluene R and heat under a V/V).
refiux condenser. with constant agitation for 90 mino Allow to Application 10 ¡lL.
coolto 60 oC and add 120 mL of methanol R to the fiask Development A Over a path of 13 cm with mobile
with constant stirring. Filter the solution through a sintered- phase A.
glass filter (16) (2. 1.2) . Rinse the fiask and the filter with Drying A In airo
25 mL of a mixture of 40 mL of toluene R and 60 mL of
Development B Over a path of 10 cm with mobile
methanol R, add the rinsing mixture to the filtrate and dilute
phase B.
10 250 mL with the same mixture of solvents.
Drying B In airo
Solution S2 Use within 4 h of preparation. Place 25 g in a
borosilicate-glass fiask with a ground-glass neck. Detection Spray with a 40 giL solution of
Add 500 mL of water for injections R and boil under a refiux phosphomolybdic acid R in anhydrous ethanol R and heat
condenser for 5 h. Allow 10 cool and decanto Reserve a in an oven at 120 cC until spots appear.
S2 and filter the rest through a sintered-glass filter (16)
(2.1.2).
2014 Appendix XX F V-AS71
Limit Any spots corresponding to plastic additive 21 Limits:

or plastic additive 20 in the chromatogram obtained - the chromatogram obtained with test solution (b) shows
with the test solution are not more intense than the only principal peaks corresponding to the peaks in the
spots in the chromatograms obtained with reference chromatogram obtained with reference solution (b) with a
solutions (b) and (c) respectively. retention time greater than 3 min;
Phenolic antioxidants Liquid chromatography (2.2.29). - the areas of the peaks in the chromatogram obtained with
Solvent mixture aeetonitrile R, tetrahydrofuran R (50:50 test solution (b) are not greater than those of the
V /V) . corresponding peaks in the chromatogram obtained with
Test solution (a) Evaporate 50 mL of solution SI to reference solution (b) .
dryness in vaeuo at 45 oC and dissolve the residue in 5.0 mL Substances soluble in hexane Place 5 g in a borosilicate-
of the solvent mixture. glass f1ask with a ground-glass neck. Add 50 mL of
Test solution (b) Evaporate 50 mL of solution SI to hexane R, fit a condenser and boil under reflux on a water-
dryness in vaeuo at 45 oC and dissolve the residue in 5.0 mL bath with constant stirring for 4 h. Cool in iced-water; a gel
of methylene ehlonde R. may formo Adapt a cooling jacket filled with iced water to a
sintered-glass filter (16) (2.1.2) fitted with a device allowing
Reference solution (a) Dissolve 25 mg of
pressure to be applied during filtration. Allow the filter to
butylhydroxytoluene CRS (plastic additive 07), 40 mg of plastie
cool for 15 min o Filter the hexane solution applying a gauge
additive 10 CRS, 40 mg of plastie additive 09 CRS and 40 mg
pressure of 27 kPa and without washing the residue;
of plastie additive 11 CRS in 10 mL of the solvent mixture.
the filtration time must not exceed 5 mino Evaporate 20 mL
Dilute 2 mL of this solution to 50.0 mL with the solvent
of the solution to dryness on a water-bath. Dry at 100 oC for
mixture.
1 h. The mas s of the residue is not greater than 40 mg
Reference solution (b) Dissolve 40 mg of plastic additive (2 per cent) if copolymer is used for containers and not
11 CRS and 40 mg of plastie additive 12 CRS in 10 mL of greater than 0.1 g (5 per cent) if copolymer is used for
methylene ehloride R. Dilute 2 mL of this solution to 50.0 mL tubing.
with methylene ehloride R.
Sulfated ash (2.4.14) Maximum 1.2 per cent, determined
Column: on 5.0 g.
- size: l = 0.25 m; 0 = 4.6 mm;
- stationalY phase: oetadeeylsi1y1 silica gel for ehromatography R Assay
(5 ¡.¡m). Introduce 0.250 g to 1.000 g of the substance to be
Mobile phase water R, tetrahydrofuran R, aeetonitrile R examined, according to the vinyl acetate content of the
(10:30 :60 VIV/V). copolymer to be examined, into a 300 mL conical f1ask with
Flow rate 1.5 mUmin. a ground-glass neck containing a magnetic stirrer.
Add 40 mL of xylene R. Boil under a reflux condenser with
Detection Spectrophotometer at 280 nm.
stirring for 4 h. Stirring continuously, allow to cool until
Injection 20 ¡.¡L of test solution (a) and reference precipitation begins before slowly adding 25.0 mL of aleoholie
solution (a). potassium hydroxide solution Rl . Boil again under a reflux
System suitability Reference solution (a): condenser with stirring for 3 h . Allow to cool with continued
- resolution: minimum 2.0 between the peaks due to plastic stirring, rinse the condenser with 50 mL of water R and add
additive 09 and plastic additive 10; 30.0 mL of 0.05 M sulfurie acid to the f1ask. Transfer the
- number of theoretieal plates: minimum 2500, ca1culated for contents of the f1ask into a 400 mL beaker; rinse the f1ask
the peak due to plastic additive 07. with two quantities, each of 50 mL, of a 200 gIL solution of
anhydrous sodium sulfate R and three quantities, each of
Limits:
20 mL, of water R and add all the rinsings to the beaker
- the chromatogram obtained with test solution (a) shows containing the initial solution. Titrate the excess sulfuric acid
only principal peaks corresponding to the peaks in the with 0.1 M sodium hydroxide, determining the end-point
chromatogram obtained with reference solution (a) with a potentiometrically (2.2.20) . Carry out a blank titration.
retention time greater than 2 min;
1 mL of 0.05 M sulfuric aeid is equivalent to 8.609 mg of
- the areas of the peaks in the chromatogram obtained with vinyl acetate.
test solution (a) are not greater than those of the
corresponding peaks in the chromatogram obtained with
reference solution (a), except for the last peak eluted in
the chromatogram obtained with reference solution (a). F. Silicone
If the chromatogram obtained with test solution (a) shows a
peak with the same retention time as the last antioxidant 1. Silicone Oi! Used as a Lubricant
eluted from reference solution (a), carry out the test as (Ph. Eur. method 3.1.8)
described with the following modifications.
Definition
Mobile phase water R, 2-propanol R, methanol R (5:45:50
Silicone oil used as a lubricant is a poly(dimethylsiloxane)
VIV/V).
obtained by hydrolysis and polycondensation of
Injection 20 ¡.¡L of test solution (b) and reference dichlorodimethylsilane and chlorotrimethylsilane . Different
solution (b) . grades exist which are characterised by a number indicating
System suitability Reference solution (b): the nominal viscosity placed after the name.
- resolution: minimum 2.0 between the peaks due to plastic Silicone oils used as lubricants have a degree of
additive 11 and plastic additive 12. polymerisation (n = 400 to 1200) such that their kinematic
visco sities are nominally between 1000 mm 2 s- 1 and
30000 mm 2 s - 1.
V-A572 Appendix XX F 2014
Characters 2. Silicone Elastomer for Closures and Tubing

Appearance Clear, colourless liquid of various viscosities . (Ph. Eur. method 3.1. 9)
Solubility Practically insoluble in water and in metbanol, Definition
very slightly soluble in anhydrous etbanol, miscible witb etbyl
Silicone elastomer complying with tbe following requirements
acetate, witb metbyl etbyl ketone and witb toluene.
is suitable for the manufacture of c10sures and tubing.
1dentification Silicone elastomer is obtained by cross-linking a linear
A. Kinematic viscosity at 25 cC (see Tests). polysiloxane constructed mainly of dimethylsiloxy units with
B. Infrared absorption spectrophotometry (2.2.24). small quantities of methylvinylsiloxy groups; the chain ends
Comparison silicone oil CRS. are blocked by trimethylsiloxy or dimetbylvinylsiloxy groups.
The region of the spectrurn from 850-750 cm - 1 is not The general formula of tbe polysiloxane is:
taken into account sin ce it may show slight differences
depending on tbe degree of polymerisation.
e. Heat 0.5 g in a test-tube over a small flame until white
fumes begin to appear. Invert the tube over a 2nd tube
containing 1 mL of a 1 gIL solution of chromotropic acid,
sodium salt R in sulfuric acid R so tbat the fumes reach
H3C, ,CH 3
tbe solution. Shake tbe 2nd tube for about lOs and heat
/Si~CH2
on a water-batb for 5 mino The solution is violet.
D. In a platinum crucible, prepare tbe sulfated ash (2.4.14)
using 50 mg. The white powder obtained gives the The cross-linking is carried out in the hot state:
reaction of silicates (2.3.1) . - either with:
Tests - 2,4-dichlorobenzoyl peroxide for extruded products; or
Acidity To 2.0 g add 25 mL of a mixture of equal - 2,4-dichlorobenzoyl peroxide or dicumyl peroxide or
volumes of anhydrous ethanol R and úher R, previously OO-(1,l-dimethyletbyl) O-isopropyl
neutralised to 0.2 mL of bromothymol blue solution Rl, and monoperoxycarbonate or 2,5-bis[(1,1-
shake. Not more tban 0.15 mL of 0. 01 M sodium hydroxide is dirnethylethyl)dioxy)-2,5-dimetbylhexane for moulded
required to change the colour of tbe solution to blue. products;
Viscosity (2.2.10) Determine tbe dynamic viscosity at - or by hydrosilylation by means of polysiloxane with -SiH
25 0e. Calculate the kinematic viscosity taking the relative groups using platinum as a catalyst.
density to be 0.97. The kinematic viscosity is witbin tbe
In all cases, appropriate additives are used such as silica and
range 95 per cent to 105 per cent of the nominal viscosity
sometirnes small quantities of organosilicon additives (cx,w-
stated on tbe label.
dihydroxypolydimethylsiloxane) .
Mineral oils Place 2 mL in a test-tube and examine in
ultraviolet light at 365 nm. The fluorescence is not more Characters
intense tban tbat of a solution containing 0.1 ppm of quinine Appearance Transparent or translucent material.
sulfate R in 0.005 M sulfuric aeid examined in tbe same
conditions. Solubility Practically insoluble in organic solvents, sorne of
which, for example cyc1ohexane, hexane and metbylene
Phenylated compounds The refractive index (2.2.6) is chloride, cause a reversible swelling of the material.
not greater than 1.410.
Heavy metals Maximum 5 ppm. 1dentification
A. Infrared absorption spectrophotometry (2.2.24) by tbe
Solvent mixture dilute ammonia R2, 2 giL solution of
multiple reflection metbod for solids.
hydroxylamine hydroehloride R (1: 9 V/V).
Comparison silieone elastomer CRS.
Mix 1.0 g with methylene ehloride R and dilute to 20 mL witb
the same .solvent. Add 1.0 mL of a freshly prepared 0.02 gIL B. Heat 1.0 g in a test-tube over a small flame until white
solution of dithizone R in methylene ehloride R, 0.5 mL of fumes begin to appear. Invert tbe tube over a 2nd tube
water R and 0.5 mL of tbe solvent mixture. At tbe same containing 1 mL of a 1 gIL solution of ehromotropie acid,
time, prepare the reference solution as follows: to 20 mL of sodium salt R in sulfurie acid R so that the fumes reach
methylene chloride R add 1.0 mL of a freshly prepared tbe solution. Shake the 2nd tube for about 10 s and heat
0.02 gIL solution of dithiz one R in methylene ehloride R, on a water-batb for 5 min. The solution is violet.
0.5 mL of lead standard solution (JO ppm Pb) R and 0.5 mL e. 50 mg of tbe residue of combustion gives the reaction of
of tbe solvent mixture. Immediately shake each solution silicates (2.3.1).
vigorously for 1 min. Any red colour in tbe test solution is Tests
not more intense than tbat in the reference solution.
Jf neeessary, cut the material into pieees of maximum dimension
Volatile matter Maxirnum 2.0 per cent, determined on on a side of not greater than 1 cm.
2.00 g by heating in an oven at 150 oC for 24 h. Carry out
Solution S Place 25 g in a borosilicate-glass flask witb a
the test using a dish 60 mm in diameter and 10 mm deep.
ground-glass neck. Add 500 mL of water R and boil under a
Labelling reflux condenser for 5 h. Allow to cool and decant the
The label states: solution.
- tbe nominal viscosity by a number placed after tbe name Appearance of solution Solution S is c1ear (2.2.1) .
of tbe product;
- that tbe contents are to be used as a lubricant.
2014 Appendix XX G V-A573
Acidity or alkalinity To 100 mL of solution S add Platinum Maximum 30 ppm.

0.15 mL of bromothymol blue solution R1 . Not more than In a quartz crucible, ignite 1.0 g of the material to be
2.5 mL of 0.01 M sodium hydroxide is required to change the examined, raising the temperature gradually until a white
colour of the indicator to blue. To a further 100 mL of residue is obtained. Transfer the residue to a graphite
solution S, add 0.2 mL of methyl orange solution R. Not more crueible. To the quartz crucible add 10 mL of a freshly
than 1. O mL of O. 01 M hydrochloric acid is required to reach prepared mixture of 1 volume of nitric acid R and 3 volumes
the beginning of the colour change of the indicator from of hydrochloric acid R, heat on a water-bath for 1-2 min and
yellow to orange. transfer to the graphite crucible . Add 5 mg of potassium
Relative density (2.2.5) 1.05 to 1.25, determined using a chloride R and 5 mL of hydrofiuoric acid R and evaporate to
density bottle with anhydrous ethanol R as the irnmersion dryness on a water-bath. Add 5 mL of hydrofiuoric acid R and
liquido evaporate to dryness again; repeat this operation twice.
Reducing substances To 20 mL of solution S add 1 mL Dissolve the residue in 5 mL of 1 M hydrochloric acid,
of dilUle sulfuric acid R and 20 mL of 0.002 M pOlassium warming on a water-bath. Allow to cool and add the solution
permanganate. Allow to stand for 15 mino Add 1 g of to 1 mL of a 250 gIL solution of stannous chlorzde R in 1 M
potassium iodide R and titrate irnmediately with O. 01 M hydrochloric acid, rinse the graphite crucible with a few
sodium thiosulfate using 0.25 mL of starch solution R as millilitres of 1 M hydrochloric acid and dilute to 10.0 mL with
indicator. Carry out a blank titration using 20 mL of water R the same acid.
instead of solution S. The difference between the titration Prepare simultaneously the reference solution as follows: to
volumes is not more than 1.0 mL. 1 mL of a 250 gIL solution of stannous chloride R in 1 M
Substances soluble in hexane Maximum 3 per cent. hydrochloric acid, add 1.0 mL of platinum standard solution
(30 ppm Pt) R and dilute to 10.0 mL with 1 M hydrochloric
Evaporate 25 mL of the solution obtained in the test for
phenylated compounds in a glass evaporating dish on a acid.
water-bath and dry in an oven at 100-105 oC for 1 h. The colour of the test solution is not more intense than that
The residue weighs not more than 15 mg. of the standard.
Phenylated compounds Place 2.0 g in a borosilicate-glass Labelling
fiask with a ground-glass neck and add 100 mL of hexane R . The label states whether the material was prepared using
Boil under a refiux condenser for 4 h. Cool, then filter peroxides or platinum.
rapidly through a sintered-glass filter (1 6) (2. 1.2). Colleet the
filtrate and close the container immediately to avoid
evaporation. At wavelengths from 250 nm to 340 nm, the
absorbance (2.2.25) is not greater than 0.4.
G. Plastic Additives
Mineral oils Place 2 g in a 100 mL conical fiask (Ph. Eur. method 3. 1.13)
containing 30 mL of a mixture of 5 volumes of ammonia R NOTE: the nomenclature given first is according ro the IUPAC
and 95 volumes of pyridine R. Allow to stand for 2 h, rules. The synonym given in bold corresponds ro the name given in
shaking frequently. Decant the pyridine solution and the texts of Chapte/" 3. The synonym corresponding ro the rules of
examine in ultraviolet light at 365 nm. The fiuorescence is the texts of "Chemical Abstracts" is also given.
not greater than that of a solution containing 1 ppm of addOl CZ4H 3S0 4. [117-81-7). PM RN 74640.
quinine sulfate R in 0.005 M sulfuric acid examined in the
same conditions.
Volatile matter Maximum 0.5 per cent for silicone
elastomer prepared using peroxides; maximum 2.0 per cent
for silicone elastomer prepared using platinum.
Weigh 10.0 g of the substance previously stored for 48 h in a
desiccator over anhydrous calcium chloride R . H eat in an oven
at 200 oC for 4 h, allow to cool in a desiccator and weigh (2RS)-2-ethylhexyl benzene-1,2-dicarboxylate
again.
synonyms: - di(2-ethylhexy1) phthalate,
Silicone elasromer prepared using peroxides complies with the - 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl)
follo wing additional test: ester.
Residual peroxides Maximum 0.08 per cent calculated as
dichlorobenzoyl peroxide .
Place 5 g in a borosilicate-glass fiask, add 150 mL of
methylene chloride R and close the fiask. Stir with a
mechanical stirrer for 16 h. Filter rapidly, collecting the
filtra te in a fiask with a ground-glass neck. Replace the air in
the container with oxygen-free nitrogen R , introduce 1 mL of a
200 gIL solution of sodium iodide R in anhydrous acetic acid R,
close the fiask, shake thoroughly and allow to stand protected zinc (2RS)-2-ethylhexanoate
from light for 30 mino Add 50 mL of water R and titrate synonyms: - zinc octanoate,
irnmediately with 0.01 M sodium thiosulfate, using 0.25 mL of - 2-ethylhexanoie acid, zinc salt (2: 1),
starch solution R as indicator. Carry out a blank titration. - zinc 2-ethylcaproate .
The difference between the titration volumes is not greater
than 2.0 mL.
Silicone elasromer prepared using platinum complies with the
following additional test:
V-A574 Appendix XX G 2014
add03 [05518-18-3)/ [00110-30-5). PM RN 53440/53520. add09 C 73 H lOS 012' [6683-19-8] . PM RN 71680.
\O~OR R"
RO.../J
N,N'-ethylenedialcanamide (with n and m = 14 or 16) OR
synonyms: - N,N'-diacylethylenediamines,
- N,N'-diacylethylenediamine (in this context
methanetetryltetramethyl tetrakis [3- [3,5-bis
acyl means in particular palmitoyl and stearoyl).
(1, l-dimethylethyl)-4-hydroxyphenyl]propanoate]
add04 [8013-07-8) . PM RN 88640. synonyrns: - pentaerythrityI tetrakis[3-(3,5-di-tert-
epoxidised soya oi! butyI-4-hydroxyphenyI)propionate] ,
add05 [8016-11-3) . PM RN 64240. - 2,2-bis [[[3-[3,5-bis(1, l-dimethylethyl)-4-
epoxidised linseed oil hydroxyphenyl] propanoyl] oxy] methyl)propane-l ,-
3-diyl 3-[3,5-bis(I,I-dimethylethyl)-4-
add06 [57455-37-5) (TSCA)/[101357-30-6]
hydroxyphenyl]propanoate,
(ElNECS)/Pigment blue 29 (Cl 77007)
- benzenepropanoic acid, 3,5-bis
ultramarine blue (1,1-dimethylethyl)-4-hydroxy-2,2-bis
add07 C 1sH 24 0 . [128-37-0) PM RN 46640. (hydroxymethyl)propane-l,3-diol ester (4: 1),
- 2,2-bis(hydroxymethyl)propane-l ,3-diol
tetrakis [3-(3,5-di-tert-butyl-4-hydroxyphenyl)
propionate ).
addlO C s4 H 78 0 3. [1709-70-2] . PM RN 95200 .
2,6-bis(l,I-dimethylethyl)-4-methylphenol R-
synonyms: - butylhydroxytoluene,
- 2,6-bis(l, l-dimethylethyl)-4-methylphenol,
- 2, 6-di -tert-butyl-4-methylphenol.
add08 CSOH660S' [32509-66-3) . PM RN 53670. 4,4' ,4"- [(2,4,6-trimethylbenzene-l ,3,5-
triyl)tris (methylene)] tris [2, 6-bis( 1, 1-dimethylethyl)phenol)
CH 3 synonyms: - 2,2' ,2" ,6,6',6 "-hexa-tert-butyI-4,4',4 "-
CH 3
H3C [ (2 ,4,6-trimethyI- 1,3 ,5-benzenetriyI)
OH
HO H3C trismethylene ]triphenoI,
- 1,3,5-tris[3,5-di-tert-butyl-4-hydroxybenzyl)-
2,4,6-trimethylbenzene,
CH 3 0 - phenol,4,4' ,4"-[(2,4,6-trimethyl-l,3,5-
benzenetriyl)tris (methylene») tris [2,6-bis
CH 3 OH (1,l-dimethylethyl)-.
CH 3 addll C3sH6203 ' [2082-79-3). PM RN 68320 .
H3C
CH 3
ethylene bis [3,3-bis [3-( 1, 1-dimethylethyl)-4- H3C~CH;"

O ./'.U CH3
hydroxyphenyl] butano ate] H3 C l· O 1 116
synonyms: - ethylene bis[3,3-bis[3-(1,1-dimethylethyI)- HO ~

4-hydroxyphenyI]butanoate], CH 3
- butanoic acid, 3,3-bis[3-(1,I-dimethylethyl)-4- H3C CH
3
hydroxyphenyl]-, 1,2-ethanediyl ester,
- ethylene bis[3,3-bis(3-tert-butyl-4-
octadecyl 3-[3,5-bis(1,1-dimethylethyl)-4-
hydroxyphenyl) butyrate].
hydroxyphenyl]propanoate
synonyms: - octadecyI 3-(3,5-di-tert-butyI-4-
hydroxyphenyI)propionate,
- propanoic acid, 3-[3,5-bis(1,1-dimethylethyl)-
4-hydroxyphenyl]-, octadecyl ester.
2014 Appendix XX G V-A575
add16 C30Hss04S. [123-28-4]. PM RN 93120.
H3C CH 3 H3C CH 3
CH3 H3C~CH3
H3C
H3C I
""" O~ I
didodeeyl 3,3' -sulfanediyldipropanoate
synonyms: - didodecyl 3,3' -thiodipropionate,
- didodeeyl 3,3' -sulfanediyldipropanoate,
H3C CH 3 I CH 3
# - propanoie aeid, 3,3' -thiobis-, dodeeyl diester,
- lauryl thiodipropionate.
CH 3
H3C CH add17 C42Hs204S, [693-36-7] . PM RN 93280.
3
tris [2,4-bis( 1, 1-dimethylethyl)phenyl] phosphite

synonyms : - tris(2,4-di-tert-buty1phenyl) phosphite,
- phenol, 2,4-bis(l,l-dimethylethyl)-,
phosphite (3:1), dioetadeeyl 3,3'-sulfanediyldipropanoate
- 2,4-bis(1, 1-dimethylethyl)phenyl, phosphite.
synonyms: - dioctadecyl 3,3' -thiodipropionate,
add13 C4sH69N306' [27676-62-6]. PM RN 95360. - dioetadeeyl 3,3'-sulfanediyldipropanoate,
- propanoie aeid, 3,3'-thiobis-,
oetadeeyl diester,
- stearyl thiodipropionate.
R- add18 [119345-01-6]. PM RN 92560.

mixture of seven produets eorresponding to
reaetion produet of di-tert-butyl phosphonite with
biphosphorous
triehloride, reaction produets with biphenyl and
1,3,S-tris [3,5-bis(l, 1-dimethylethyl)-4-hydroxybenzyl]-1 ,3,5- 2,4-bis( 1, 1-dimethylethyl)phenol:
triazine-2,4,6(lH,3H,SH)-trione
synonyms: - 1,3,5-tris(3,5-di-tert-butyl-4-
hydroxybenzy1)-s-triazine-2,4,6(lH,3H,5H)- R./ H3C~CH3
(1(
trione,
- 1,3,S-triazine-2,4,6(1H,3H,SH)-trione, H3C CH 3 H3C CH 3
1,3,5-tris [[3,5-bis( 1, 1-dimethylethyl)-4-hydroxy-

phenyl]methyl]-. component 1
add14 C41Hs206P2' [3806-34-6]. PM RN 50080.
2,4-bis(1, 1-dimethylethyI)phenyI biphenyI-4,4'-

diyldiphosphonite
component JI
3,9-bis(oetadeeyloxy)-2,4,8, 1O-tetraoxa-3,9-
diphosphaspiro [5.5] undeeane RO
\
P-OR
synonyms: - 2,2'-bis(octadecyloxy)-5,5'-spirobi
[1 ,3,2-dioxaphosphinanel, R\ ' /\ rl
- 2,4,8,10-tetraoxa-3,9-
diphosphaspiro [5.5] undeeane, 3, 9-bis
RÓ~
(oetadeeyloxy)-. 2,4-bis(1,1-dimethylethyI)phenyI biphenyI-3,4'-
add15 C36H74S2' [2500-88-1]. PM RN 49840 . diyldiphosphonite
component JII
S
I
S RO
\
P-OR
1,1 '-disulfanediyldioetadeeane ~
synonyms: - dioctadecyl disulfide, }J\J
- oetadeeane, 1,1 '-dithio-. RO-P\
OR
2,4-bis(1,1-dimethylethyI)phenyI biphenyI-3,3'-
diyldiphosphonite
component IV
V-A576 Appendix XX H 2014
add23
mixture of component 1 and about 27 per cent of component
II
2,4-bis(l,1-dimethylethyl)phenyl biphenyl-4-ylphosphonite component I [26401-97-8]
component V
OR R-
I
RO-P,
OR
2,4-bis(l,1-dimethylethyl)phenyl phosphite bis [(2RS)-2-ethylhexyl]

2,2'- [( dioctylsta=anetriyl)bis(sulfanediyl)] diacetate
component VI
synonyms: - di(isooctyI) 2,2 '-
[(dioctyIstannylene)bis(thio) ]diacetate,
- bis (isooctyloxycarbonylmethylthio)
dioctylsta=ane.
component II [26401-86-5]
2,4-bis(1,1-dimethylethyl)phenyl 4'-[bis [2,4-bis(1, 1-
dimethylethyl)phenoxy] phosphanyl] biphenyl-4-ylphosphona te
component VII
R-OH: 2,4-bis (1, 1-dimethylethyl)phenol
add19 C ls H 360 2' [57-11-4]. PM RN 24550.
tris[(2RS)-2-ethylhexyl] 2,2',2"-
[(octylsta=anetriyl) tris (sulfanediyl)] triacetate
synonyms: - tri(isooctyI) 2,2 ' ,2 " -
octadecanoic acid [(monooctyIstannylidyne)tris(thio) ]
synonyms: - stearic acid, triacetate,
- octadecanoic acid. - 2,2',2'1_[( octylsta=ylidyne)tris(thio)]triacetic
acid, triisooctyl estero
add20 C 1s H 3S NO. [301-02-0] . PM RN 68960.
H. Polyethylene Terephthalate for

Containers for Preparations not for
(Z)-octadec-9-enamide
Parenteral Use
synonyms: - oIeamide,
- 9-octadecenamide, (Z) -, (Ph. Eur. method 3.1.15)
- 9-cis-okamide.
add21 C 22 H 43 NO. [112-84-5]. PM RN 52720
r ~ O"OO~"
io~O~ J
(Z) -docos-13-enamide Definition
synonyms: - erucamide, Polyethylene terephthalate is obtained from the
- 13-docosenamide, (Z) -, polymerisation of terephthalic acid or dimethyl terephthalate
,-- 13-cÍ5-docosenamide. with ethylene glycol. Isophthalic acid, dimethyl isophthalate,
add22 [65447-77-0]. PM RN 60800 l,4-bis(hydroxymethyl)cyclohexane (cyclohexane-1 ,4-
dimethanol) or diethylene glycol may be used in the
polymerisation. It may contain not more than 0.5 per cent of
silica or silicates and colouring matter approved by the
competent authority.
Production
The manufacturing pro ces s is validated to demonstrate that
the residual acetaldehyde content is not greater than 10 ppm
copolymer of dimethyl butanedioate and 1-(2-hydroxyethyl)- in the granules.
2,2,6,6-tetramethylpiperidin-4-ol
Characters
synonyms: - copolymer of dimethyI succinate and
(4-hydroxy- 2,2 ,6,6-tetramethylpiperidin-l-y- Appearance clear or opaque granules.
I)ethanol. SoIubility practically insoluble in water, in ethanol
(96 per cent) and in methylene chloride. It is hydrolysed by
strong bases .
2014 Appendix XX H V-AS77
Identification Reducing substances Add 2 mL of 0.5 M sulfuric acid

A. Place 0.10 g of the material to be examined into a and 20.0 mL of 0.002 M potassium permanganate ro 20.0 mL
borosilicate glass ftask with a ground-glass neck. of solution SI. Boil for 3 mino Cool immediately ro ambient
Add 25 mL of a 200 giL solution of potassium temperature. Add 1 g of potassium iodide R, 0.25 mL of
hydroxide R in a 50 per cent VIV solution of anhydrous starch solution R as indicator and titrate with 0.01 M sodium
ethanol R. Reftux for 30 mino Allow to cool and dilute ro thiosulfate. Perform a blank titration using 20.0 mL of
100 mL with water R. Filter ifnecessary. Dilute 1.0 mL water R. The difference in volume used in the 2 titrations is
of the filtra te to 100 mL with water R. Examined not greater than 0.5 mL.
between 210 nm and 330 nm (2.2.25), the solution Substances soluble in dioxan MaxÍmum 3 per cent.
shows an absorption maximum at 240 nm. Place 2 g of the material to be examined in a borosilicate
B. Dissolve 0.05 g of the material to be examÍned in 2 mL glass ftask with a ground-g1ass neck. Add 20 mL of dioxan R
of 1,1,1,3,3,3-hexafiuoropropan-2-o1 R. Apply to a glass and heat under reftux for 2 h. Evaporate 10 mL of the
plate on a water-bath in a fume-cupboard several drops solution ro dryness on a water-bath and then dry the residue
of the solution to produce a film of about 15 mm by at 100-105 oC . The residue weighs a maxÍmum of 30 mg.
15 mm. Allow the solvent to evaporate and remove the Extractable aluminium Maximum 1 ppm.
film using a stream of water and a scraper. Dry in an Inductively coupled plasma-atomic emission spectrometry
oven at 100-105 oC for 1-2 h. Examine the film by (2.2.57).
infrared absorption spectrophotometry (2.2.24).
The spectrum of the material to be examined shows Test solution Solution S3.
maxima in particular at 1725 cm- l , 1410 cm- l, Reference solutions Prepare the reference solutions using
1265 cm- l, 11 20 cm- l, 1100 cm- \ 1020 cm- l, aluminium standard solution (200 ppm Al) R, diluting with
875 cm- l, 725 cm- l. The spectrum obtained, in 0.1 M hydrochloric acid.
addition, is identical to that of the material selected for Wavelength 396.15 nm, the spectral background being
the type sample. taken at 396.25 nm.
Tests Verify the absence of aluminium in the 0.1 M hydrochloric
lf necessaJY,cut out samples for testing to a maximum size of acid used.
1 cm per side. Extractable antimony Maximum 1 ppm.
Solution SI Place 10.0 g of the material ro be examined Inductively coupled plasma-aromic emission spectrometry
in a borosilicate glass ftask with a ground-glass neck. (2.2.57).
Add 200 mL of water R and heat at 50 oC for 5 h. Allow to Test solution Solution S4.
cool and decant the solution. Use solution Sl within 4 h of its Reference solutions Prepare the reference solutions using
preparation. antimony standard solution (100 ppm Sb) R, diluting with
Solution S2 Place 10 g of the material to be examined iri 0.01 M sodium hydroxide.
a borosilicate glass ftask with a ground-glass neck. Wavelength 231.15 nm or 217.58 nm, the spectral
Add 100 mL of ethanol (96 per cent) R and heat at 50 oC for background being taken at 231.05 nm.
5 h. Allow to cool and decant the solution. Use solution S2
Extractable barium Maximum 1 ppm.
within 4 h of its preparation.
Solution S3 Place 20 g of the material to be examined in
(2.2.57).
a borosilicate glass ftask with a ground-glass neck.
Add 50 mL of 0.1 M hydrochloric acid and heat at 50 oC for Test solution Solution S3.
5 h. Allow ro cool and decant the solution. Use solution S3 Reference solutions Prepare the reference solutions using
within 4 h of its preparation. barium standard solutíon (50 ppm Ea) R , diluting with 0. 1 M
Solution S4 Place 20 g of the material to be examined hydrochloric acid.
into a borosilicate glass ftask with a ground-glass neck. Wavelength 455.40 nm, the spectral background being
Add 50 mL of 0.01 M sodium hydroxide and heat at 50 oC taken at 455.30 nm.
for 5 h. Allow to cool and decanto Use solution S4 within 4 h Verify the absence of barium in the 0.1 M hydrochloric acid
of its preparation. used.
Appearance ofsolution SI Solution SI is c1ear (2.2. 1) . Extractable cobalt Maximum 1 ppm.
Appearance of solution S2 Solution S2 is c1ear (2.2.1) Inductively coupled plasma-atomic emission spectrometry
and colourless (2.2.2, Method 11). (2.2.57).
Acidity or aIkalinity To 50 mL of solution SI add Test solution Solution S3.
0.15 mL of ERP indicator solution R. The solution tums Reference solutions Prepare the reference solutions using
yellow. Not more than 0.5 mL of 0.01 M sodium hydroxide is cobalt standard solution (100 ppm Co) R , diluting with 0.1 M
required to change the colour of the indicator to blue. hydrochloric acid.
To another 50 mL of solution SI add 0.2 mL of methyl
Wavelength 228 .62 nm, the spectral background being
orange solution R. The solution tums yellow. Not more than
taken at 228.50 nm.
0.5 mL of O. 01 M hydrochloric acid is required to reach the
beginning of the colour change of the indicator ro orange. Verify the absence of cobalt in the 0.1 M hydrochloric acid
used.
Absorbance of solution SI (2.2.25) Maximum 0.20
between 220 nm and 340 nm. In addition, for coloured Extractable germanium Maximum 1 ppm.
polyethylene terephthalate: maximum 0.05 between 400 nm Inductively coupled plasma-atomic emission spectrometry
to 800 nm. (2.2.57) .
Absorbance of solution S2 (2.2.25) MaxÍmum 0.05 Test solution Solution S4.
between 400 nm and 800 nm.
V-A578 Appendix XX H 2014

germanium standard solution (100 ppm Ge) R, diluting with
0.01 M sodium hydroxide.
Wavelength 206.87 nm or 265.12 nm, the spectral
background being taken at 206.75 nm.
Extractable manganese Maximum 1 ppm.
(2.2.57) .
Test solution Solution S3.
manganese standard solution (100 ppm Mn) R, diluting with
0. 1 M hydrochloric acid.
Wavelength 257.61 nm, the spectral background being
taken at 257.50 nm.
Verify the absence of manganese in the 0.1 M hydrochloric
acid used.
Extractable titanium Maximum 1 ppm.
(2.2.57) .
titanium standard solution (100 ppm TI) R, diluting with
0. 1 M hydrochloric acid.
Wavelength 323.45 nm or 334.94 nm, the spectral
background being taken at 323.35 nm.
Verify the absence of titanium in the O. 1M hydrochloric acid
used.
Extractable zinc Maximum 1 ppm.
(2.2.57).
z inc standard solution (100 ppm Zn) R, diluting with 0. 1 M
hydrochloric acid.
Wavelength 213.86 nm, the spectral background being
taken at 213.75 nm.
Verify the absence of zinc in the 0.1 M hydrochloric acid used.
Sulfated ash (2.4.14) Maximum 0.5 per cent determined
on 1.0 g.
2014 Appendix XXI B V-A579
Preparations
Appendix XXI Applic.
Caps.
Application
Capsules
Ext. Extract
A. Abbreviated Titles Inf. Infusion
In accordance with the General Notices the main title of Inj . Injection
each monograph may be abbreviated using the following Lin. Liniment
abbreviations. An abbreviated title has the same significance Lot. Lotion
as the main title. Mixt. Mixture
Cations Oint. Ointment
Pess. Pessaries
Alum. Aluminium
Soln. Solution
Arnmon. Ammonium
Suppos. Suppositories
Bism. Bismuth
Tabs. Tablets
Calco Calcium
Tinct. Tincture
Ferr. Ferrous
Mag. Magnesium
Pot. Potassium
Sod. Sodium B. Approved Synonyms
Where the English title at the head of a monograph in the
Anions European Pharrnacopoeia is different from that at the head of
Acet. Acetate the text incorporated into the British Pharrnacopoeia or the
Benz. Benzoate British Pharrnacopoeia (Veterinary), an Approved Synonym
Brom. Bromide (or Approved Synonyms) is created on the recommendation
Chlor. Chloride of the British Pharrnacopoeia Commission.
Cit. Citrate In accordance with the General Notice on Titles, the name
Dihydrochlor. Dihydrochloride or names given in the right-hand column of the list below are
Fumar. Fumarate Approved Synonyms for the name at the head of the
Hydrobrom. Hydrobromide monograph of the European Pharrnacopoeia given in the
Hydrochlor. H ydrochloride left-hand column. Where there is more than one entry in
Hydrox. Hydroxide the right-hand column, the first entry is used as the title of
Iod. Iodide the monograph in the British Pharrnacopoeia or the British
Lact. Lactate Pharrnacopoeia (Veterinary) and the remaining entries are
Mal. Maleate included as subsidiary titles.
Methonit. Methonitrate
Approved Synonyms and subsidiary titles have the same
Methylsulf. Methylsulfate
significance as the main title and are thus official titles.
Metilsulf. Metilsulfate
Nit. Nitrate Names made by changing the order of the words in an
Ox. Oxide Approved Synonym, with the addition of a preposition when
Phenylprop. Phenylpropionate necessary, are also Approved Synonyms.
Phos. Phosphate Where square brackets are used in a title these may be
Prop. Propionate replaced by round brackets, and vice versa . The words
Succino Succinate 'per cent' may be replaced by the symbol '%'.
Sulf. Sulfate Where the word 'Injection' appears in the title or synonym of
Tart. Tartrate a monograph in the European Pharrnacopoeia, the
abbreviation 'Inj.' is declared to be an Approved Synonym
Adjectives for that part of the title.
Ammon. Ammoniated Monographs included in the British Pharrnacopoeia
Arom. Aromatic (Veterinary) are identified by means of a superscript
Camph. Camphorated number: 1 .
CO. Compound
Conc. Concentrated
Cryst. Crystalline
Efferv. Effervescent
Emulsif. Emulsifying
Liq. Liquefied; Liquid
Prep. Prepared
Simp. Simple
V-AS80 Appendix XXI B 2014
EUROPEAN PHARMACOPOEIA TlTLE APPROVED SYNONYM

Medicinal Substances, Fonnulated Preparations and Herbal Drugs
~-Acetyldigoxin Acetyldigoxin
Acetylsalicylic Acid Aspirin
N-Acetyltryptophan Acetyltryptophan
N-Acetyltyrosine Acetyltyrosine
Adrenaline Tartrate Adrenaline Acid Tartrate I Epinephrine Acid Tartrate
Medicinal Air Medical Air
Synthetic Medicinal Air Synthetic Air
Hydrated Aluminium Oxide Dried Aluminium Hydroxide
Hydrated Aluminium Phosphate Dried Aluminium Phosphate
4-Aminobenzoic Acid Aminobenzoic Acid
Concentrated Ammonia Solution Strong Arnmonia Solution
Ammonium Glycyrrhizate Arnmonium Glycyrrhizinate
Ammonium Hydrogen Carbonate Ammonium Bicarbonate
Amphotericin B Amphotericin
Anhydrous Ampicillin Ampicillin
Anamirta Cocculus for Homoeopathic Preparations Cocculus Indicus for Homoeopathic Preparations
Angelica Sinensis Root Processed Angelica Sinensis Root
Azaperone for Veterinaty Use Azaperone 1
Baical Skullcap Root Scutellariae Baicalensis Root
Standardised Belladonna Leaf Tincture Belladonna Tincture
Dried Bilberry Fruit Dried Bilberry
Fresh Bilberry Fruit Fresh Bi!berty
Refined Borage (Starflower) Oil Refined Borage Oil
Refined Starflower Oi!
Butyl Parahydroxybenzoate Butyl Hydroxybenzoate
Butylparaben
Butylhydroxyanisole Butylated Hydroxyanisole
Butylhydroxytoluene Butylated Hydroxytoluene
Caffeine Monohydrate Caffeine Hydrate
Ca1cium Hydrogen Phosphate Dihydrate Ca1cium Hydrogen Phosphate
Dibasic Ca1cium Phosphate
o-Camphor Natural Camphor
Captylic Acid Octanoic Acid
Caraway Fruit Caraway
Carprofen for Veterinary Use Carprofen 1
Cefepime Dihydrochloride Monohydrate Cefepime Hydrochloride Monohydrate
Cellulose Acetate Phthalate Cellacefate
Cetirizine Di~drochloride Cetirizine Hydrochloride
Roman Chamomi!e Flower Chamomile Flowers
Chlorhexidine Diacetate Chlorhexidine Acetate
Chlorhexidine Digluconate Solution Chlorhexidine Gluconate Solution
Chlorhexidine Dihydrochloride Chlorhexidine Hydrochloride
Chlorobutanol Hemihydrate Chlorobutanol
Choleca1ciferol Coleca1ciferol
Choleca1ciferol Concentrate (Oily Form) Coleca1ciferol Concentrate (Oily Form)
Cholecalciferol Concentrate (Powder Forrn) Coleca1ciferol Concentrate (Powder Form)
EUROPEAN PHARMACOPOEIA TITLE APPROVED SYNONYM

Cholecalciferol Concentrate (Water-dispersible Forrn) Colecalciferol Concentrate (Water-dispersible Forrn)
Cisapride Monohydrate Cisapride
Clazuril for Veterinary U se Clazuril 1
Clodronate Disodium Tetrahydrate Sodium Clodronate Tetrahydrate
Closantel Sodium Dihydrate for Veterinary Use Closantel Sodium Dihydrate 1
Refined Coconut-oil Coconut Oil
Codeine Hydrochloride Dihydrate Codeine Hydrochloride
Codeine Phosphate Hemihydrate Codeine Phosphate
Narrow-Ieaved Conefiower Root Echinacea Angustifolia Root
Pale Conefiower Root Echinacea Pallida Root
Purple Conefiower Herb Echinacea Purpurea Herb
Purple Conefiower Root Echinacea Purpurea Root
Cysteine Hydrochloride Monohydrate Cysteine Hydrochloride
Deferoxamine Mesilate Desferrioxamine Mesilate
Dembrexine Hydrochloride Monohydrate for Veterinary Use Dembrexine Hydrochloride Monohydrate 1
Detomidine Hydrochloride for Veterinary Use Detomidine Hydrochloride 1
Devil's Claw Root Devil's Claw
Harpagophytum
Dibrompropamidine Diisetionate Dibrompropamidine Isetionate
Diclazuril for Veterinary Use Diclazuril 1
Difioxacin Hydrochloride Trihydrate for Veterinary Use Difioxacin Hydrochloride Trihydrate l
Dihydrocodeine Hydrogen Tartrate Dihydrocodeine Tartrate
Dihydrostreptomycin Sulfate for Veterinary Use Dihydrostreptomycin Sulfate l
Dipotassium Phosphate Dipotassium Hydrogen Phosphate
Anhydrous Disodium Phosphate Anhydrous Disodium Hydrogen Phosphate
Disodium Phosphate Dihydrate Disodium Hydrogen Phosphate Dihydrate
Sodium Phosphate Dihydrate
Disodium Phosphate Dodecahydrate Disodium Hydrogen Phosphate Dodecahydrate
Disodium Hydrogen Phosphate
Sodium Phosphate
Dopexamine Dihydrochloride Dopexamine Hydrochloride
Doxylamine Hydrogen Succinate Doxylamine Succinate
Emedastine Difumarate Emedastine Fumarate
Enilconazole for Veterinary Use Enilconazole 1
Enrofioxacin for Veterinary Use Enrofioxacin 1
Ephedrine Hemihydrate Ephedrine
Equine Serum Gonadotrophln for Veterinary Use Serum Gonadotrophin 1
Equisetum Stem Horsetail
Erythromycin Ethylsuccinate Erythromycin Ethyl Succinate
Anhydrous Ethanol Ethanol
Absolute Alcohol
Dehydrated Alcohol
Ethyl Parahydroxybenzoate Ethyl Hydroxybenzoate
Ethylparaben
Febantel for Veterinary Use Febantel 1
Fenbendazole for Veterinary Use Fenbendazole l
Flunixin Meglumine for Veterinary Use Flunixin Meglumine l
Flupentixol Dihydrochloride Flupentixol Hydrochloride
Fluphenazine Dihydrochloride Fluphenazine Hydrochloride
V-A582 Appendix XXI B 2014

Foscamet Sodium Hexahydrate Foscamet Sodium
Fourstamen Stephania Root Stephania Tetrandra Root
Gentian Root Gentian
Glucose Monohydrate Glucose
Goldenseal Rhizome Goldenseal Root
Hexamidine Diisetionate Hexamidine Isetionate
Concentrated Hydrochloric Acid Hydrochloric Acid
Hydrocodone Hydrogen Tartrate 2.5-Hydrate Hydrocodone Hydrogen Tartrate Hydrate
myo-Inositol Inositol
Insulin Zinc Injectable Suspension Insulin Zinc Suspension
Insulin Zinc Suspension (Mixed)
Insulin Zinc Injectable Suspension (Amorphous) Insulin Zinc Suspension (Amorphous)
Insulin Zinc Injectable Suspension (Crystalline) Insulin Zinc Suspension (Crystalline)
Isophane Insulin Injection Isophane Insulin
Isophane Insulin (NPH)
Soluble Insulin Injection Insulin Injection
Neutral Insulin
Neutral Insulin Injection
Soluble Insulin
Kanamycin Monosulfate Kanamycin Sulfate
Ketotifen Hydrogen Fumarate Ketotifen Fumarate
Lactose Monohydrate Lactose
Liquid Lactulose Lactulose Solution
Levamisole for Veterinary Use Levamisole l
Liquorice Root Liquorice
Low-molecular-mass Heparins Low-molecular-weight Heparins
Lufenuron (Anhydrous) for Veterinary Use Anhydrous Lufenuron I
Macrogolglycerol Hydroxystearate Hydrogenated Polyoxyl Castor Oil
Macrogolglycerol Ricinoleate Polyoxyl Castor Oil
Magnesium Aspartate Dihydrate Magnesium Aspartate
Magnesium Chloride 4.5-Hydrate Partially Hydrated Magnesium Chloride
Marbofioxacin for Veterinary Use Marbofioxacin I
Matricaria Flower Matricaria Flowers
Meclozine Dihydrochloride Meclozine Hydrochloride
Melissa Leaf Lemon Balm
Melissa Leaf Dry Extract Lemon Balm Dry Extract
Metamizole Sodium Monohydrate Dipyrone
Methyl Parahydroxybenzoate Methyl Hydroxybenzoate
Methylparaben
Methylene Chloride Dichloromethane
Methy1hydroxyethylcellulose Hydroxyethylmethylcellulose
N-Methylpyrrolidone Methylpyrrolidone
Partly Dementholised Mint Oil Dementholised Mint Oil
Morantel Hydrogen Tartrate for Veterinary Use Morantel Tartrate l
Moxidectin for Veterinary Use Moxidectin I
Naftidrofuryl Hydrogen Oxalate Naftidrofuryl Oxalate
Naloxone Hydrochloride Dihydrate Naloxone Hydrochloride
Nitrofural Nitrofurazone

Noradrenaline Hydrochloride Noradrenaline Hydroehloride / Norepinephrine
Hydroehloride
Noradrenaline Tartrate Noradrenaline Acid Tartrate / Norepinephrine Aeid Tartrate
Omega-3-aeid Triglyeerides Omega-3 Marine Triglyeerides
Raw Opium Opium
Bitter-orange Epiearp and Mesocarp Dried Bitter-Orange Peel
Bitter-orange Epiearp and Mesoearp Tineture Orange Tineture
Orbifioxaein for Veterinary U se Orbifioxaein 1
Oxfendazole for Veterinary U se Oxfendazole 1
Panereas Powder Panereatie Extraet
Wild Pansy (Flowering Aerial Parts) Wild Pansy
Pefioxaein Mesilate Dihydrate Pefioxaein Mesilate
Pentamidine Diisetionate Pentamidine Isetionate
Pentoxyverine Hydrogen Citrate Pentoxyverine Citrate
Pepsin Powder Pepsin
Perindopril tert-Butylamine Perindopril Erbumine
Coneentrated Phosphorie Acid Phosphorie Acid
Pirenzepine Dihydroehloride Monohydrate Pirenzepine Hydroehloride
Ribwort Plantain Plantain
Potassium Hydrogen Carbonate Potassium Bicarbonate
Primaquine Diphosphate Primaquine Phosphate
Propyl Parahydroxybenzoate Propyl Hydroxybenzoate
Propylparaben
Pygeum Afrieanum Bark Pygeum Bark
Racemie Ephedrine Hydroehloride Racephedrine Hydroehloride
Racemie Menthol Raeementhol
Sage Leaf (Salvia officinalis) Sage Leaf
Claty Sage Oil Sage Oil
Sanguisorba Root Greater Burnet Root
Selameetin for Veterinary Use Selamectin 1
Selenium Disulfide Selenium Sulfide
Common Selfheal Fruit-spike Selfheal Fruit-Spike
Alexandrian Senna Pods Alexandrian Senna Fruit
Tinnevelly Senna Pods Tinnevelly Senna Fruit
Sodium Hydrogen Carbonate Sodium Biearbonate
Sodium Laurilsulfate Sodium Lauryl Sulfate
Sodium Dodecyl Sulfate
Sodium Ethyl Parahydroxybenzoate Ethyl Hydroxybenzoate Sodium
Ethylparaben Sodium
Sodium Methyl Parahydroxybenzoate Sodium Methyl Hydroxybenzoate
Sodium M ethylparaben
Hydrated Sodium Perborate Sodium Perborate
Sodium Propyl Parahydroxybenzoate Sodium Propyl Hydroxybenzoate
Sodium Propylparaben
Sodium Sulfate Deeahydrate Sodium Sulfate
Glauber's Salt
Solutions for Haemofiltration and for Haemodiafilu"ation Haemofiltration and Haemodiafiltration Solutions

Hydrogenated Soya-bean Oil Hydrogenated Soya Oil
Hydrogenated Soyabean Oil
Refined Soya-bean Oil Refined Soya Oil
Refined Soyabean Oil
Spectinomycin Sulfate Tetrahydrate for Veterinary Use Spectinomycin Sulfate Tetrahydrate 1
Sulfamethoxypyridazine for Veterinary Use Sulfametoxypyridazine 1
Tale Purified Tale
Anhydrous Theophylline-Ethylenediamine Aminophylline
Theophylline- Ethylenediamine Hydrate Aminophylline Hydrate
TheophylIine Monohydrate Theophylline Hydrate
Thiopental Sodium and Sodium Carbonate Thiopental Sodium
Tiamulin for Veterinary Use Tiamulin 1
Tiamulin Hydrogen Fumarate for Veterinary Use Tiamulin Hydrogen Fumarate 1
alI -rac-C(- Tocopherol all-rac-Alpha Tocopherol
all-rac-IX-Tocopheryl Acetate all-rac-Alpha Tocopheryl Acetate
DL-IX-Tocopheryl Hydrogen Succinate Alpha Tocopheryl Hydrogen Succinate
RRR-IX- Tocopherol RRR-Alpha Tocopherol
RRR-IX-Tocop_heryl Acetate RRR-Alpha Tocopheryl Acetate
- RRR-IX- Tocopheryl Hydrogen Succinate RRR-Alpha Tocopheryl Hydrogen Succinate
Tri-n-butyl Phosphate Tributyl Phosphate
Trimetazidine Dihydrochloride Trimetazidine Hydrochloride
Trolamine Triethanolamine
Tylosin for Veterinary U se Tylosin 1
Tylosin Phosphate Bulk Solution for Veterinary Use Tylosin Phosphate 1
Tylosin Tartrate For Veterinary Use Tylosin Tartrate 1
Undecylenic Acid Undecenoic Acid
Valerian Root Valerian
Cut Valerian Root Cut Valerian
Valnemulin Hydrochloride for Veterinary Use Valnemulin Hydrochloride 1
Vedaprofen for Veterinary Use Vedaprofen 1
Synthetic Vitamin A Concentrate (Oily Form) Synthetic Retinol Concentrate (Oily Form)
Synthetic Vitamin A Concentrate (Powder Form) Synthetic Retinol Concentrate (Powder Form)
Synthetic Vitamin A Concentrate Solubilisate / Emulsion Synthetic Retinol Concentrate, (Solubilisate / Emulsion)
Synthetic Retinol Concentrate, (Water-dispersible Form)
Xylazine Hydrochloride for Veterinary Use Xylazine Hydrochloride 1
Zinc Acetate Dihydrate Zinc Acetate
Zinc Undecylenate Zinc Undecenoate

Blood-related Products
Anticoagulant and Preservative Solutions for Human Blood Anticoa~~lant and Preservative Solutions for Blood
Human Albumin Solution Alburnin Solution
Albumin
Human Albumin
Human Anti-D Immunoglobulin Anti-D (Rh o) Irnmunoglobulin
Human Anti-D Immunoglobulin for Intravenous Anti-D Immunoglobulin for Intravenous Use
Administration
Human Antithrombin III Concentrate Antithrombin III Concentrate
Human Coagulation Factor VII Dried Factor VII Fraction
Human Coagulation Factor VIII Dried Factor VIII Fraction
Human Coagulation Factor VIII (rDNA) Dried Factor VIII (rDNA)
Human Coagulation Factor IX Dried Factor IX Fraction
Human Coagulation Factor XI Dried Factor XI Fraction
Human Fibrinogen Dried Fibrinogen
Human Hepatitis A Immunoglobulin Hepatitis A Immunoglobulin
Human Hepatitis B Immunoglobulin Hepatitis B Immunoglobulin
Human Hepatitis B Immunoglobulin for Intravenous Hepatitis B Immunoglobulin for Intravenous Use
Administration
Human Measles Immunoglobulin Measles Immunoglobulin
Human Normal Immunoglobulin Normal Immunoglobulin
Normal Immunoglobulin Injection
Human Normal Immunoglobulin for Intravenous Normal Immunoglobulin for Intravenous Use
Administration
Human Plasma for Fractionation Plasma for Fractionation
Human Plasma (Pooled and Treated for Virus Inactivation) Plasma (Pooled and Treated for Virus Inactivation)
Human Prothrombin Complex Dried Prothrombin Complex
Human Rabies Irnmunoglobulin Rabies Irnmunoglobulin
Human Rubella Immunoglobulin Rubella Immunoglobulin
Human Tetanus Immunoglobulin Tetanus Immunoglobulin
Human Varicella Immunoglobulin Varicella Immunoglobulili
Human Varicella Immunoglobulin for Intravenous Varicella Immunoglobulin for Intravenous Use
Administration
Human von Willebrand Factor von Willebrand Factor

Immunological Products ~Human)
BCG Vaccine, Freeze-dried BacilIus Calmene-Guérin Vaccine
BCG Vaccine
Diphtheria and Tetanus Vaccine (Adsorbed) Adsorbed Diphtheria and Tetanus Vaccine
Diphtheria, Tetanus and Pertussis (Acellular Component) Adsorbed Diphtheria, T etanus and Pertussis (Acellular
Vaccine (Adsorbed) Component) Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Adsorbed Diphtheria, Tetanus, Pertussis (AcelIular
Haemophilus Type b Conjugate Vaccine (Adsorbed) Component) and Haemophilus Type b Conjugate Vaccine
Diphtheria, Tetanus, Pertussis (Acellular Component) and Adsorbed Diphtheria, Tetanus, Pertussis (AcelIular
Hepatitis B (rDNA) Vaccine (Adsorbed) Component) and Hepatitis B (rDNA) Vaccine
Diphtheria, Tetanus, Pertussis (AcelIular Component) and Adsorbed Diphtheria, Tetanus, Pertussis (Acellular
Poliomyelitis (Inactivated) Vaccine (Adsorbed) Component) and Inactivated Poliomyelitis Vaccine
Diphtheria Vaccine (Adsorbed) Adsorbed Diphtheria Vaccine
Gas-gangrene Antitoxin (Novyi) Gas-gangrene Antitoxin (Oedematiens)
Hepatitis A Vaccine (Inactivated, Adsorbed) Inactivated Hepatitis A Vaccine
Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine Hepatitis A (Inactivated) and Hepatitis B (rDNA) Vaccine
(Adsorbed)
Influenza Vaccine (Split Virion, Inactivated) Inactivated Influenza Vaccine (Split Virion)
Influenza Vaccine (Surface Antigen, Inactivated) Inactivated Influenza Vaccine (Surface Antigen)
Influenza Vaccine (Whole Virion, Inactivated) Inactivated Influenza Vaccine (Whole Virion)
Measles Vaccine (Live) Measles Vaccine, Live
Measles, Mumps and Rubella Vaccine (Live) Measles, Mumps and Rubella Vaccine, Live
Mum¡Js Vaccine (Live) Mumps Vaccine, Live
Tuberculin for Human Use, Old Old Tuberculin
Pertussis Vaccine (Acellular Component, Adsorbed) Adsorbed Pertussis Vaccine (Acellular Component)
Pertussis Vaccine (Acellular, Co-purified, Adsorbed) Adsorbed Pertussis Vaccine (AcelIular, Co-purified)
Poliomyelitis Vaccine (Inactivated) Inactivated Poliomyelitis Vaccine
Poliomyelitis Vaccine (Oral) Polio~yelitis Vaccine, Live (Oral)

Rabies Vaccine for Human Use Prepared in Cell Cultures Rabies Vaccine
Rubella Vaccine (Live) Rubella Vaccine, Live
Tetanus Antitoxin for Human Use Tetanus Antitoxin
Tetanus Vaccine (Adsorbed) Adsorbed Tetanus Vaccine
Tick-borne Encephalitis Vaccine (Inactivated) Tick-borne Encephalitis Vaccine, Inactivated
Tuberculin Purified Protein Derivative for Human Use Tuberculin Purified Protein Derivative
Tuberculin P.P.D.
Typhoid Vaccine (Live, Oral, Strain Ty 21a) Typhoid (Strain Ty 21a) Vaccine, Live (Oral)
Varicella Vaccine (Live) Varicella Vaccine, Live
Yellow Fever Vaccine (Live) Yellow Fever Vaccine, Live
Irnmunological Products (Veterinary)

Monographs included in the British Phannacopoeia (Veterinary)
Anthrax Spore Vaccine (Live) for Veterinary Use Anthrax Vaccine, Living
Aujeszky's Disease Vaccine (Inactivated) for Pigs Aujeszky's Disease Vaccine, Inactivated
Aujeszky's Disease Vaccine (Live) for Pigs for Parenteral Aujeszky's Disease Vaccine, Living
Administration
Avian Infectious Bronchitis Vaccine (Live) Avian Infectious Bronchitis Vaccine, Living
Avian Infectious Bursal Disease Vaccine (Inactivated) Infectious Bursal Disease Vaccine, Inactivated
Gumboro Disease Vaccine, Inactivated
Avian Infectious Bursal Disease Vaccine (Live) Infectious Bursal Disease Vaccine, Living
Gumboro Disease Vaccine, Living
Avian Infectious Encephalomyelitis Vaccine (Live) Infectious Avian Encephalomyelitis Vaccine, Living Epidemic
Tremor Vaccine, Living
Avian Infectious Laryngotracheitis Vaccine (Live) Laryngotracheitis Vaccine, Living
Bovine Parainfluenza Virus Vaccine (Live) Bovine Parainfluenza Virus Vaccine, Living
Bovine Respiratory Syncytial Virus Vaccine (Live) Bovine Respiratory Syncytial Virus Vaccine, Living
Brucellosis Vaccine (Live) (Brucella Melitensis Rev. 1 Strain) Brucella Melitensis (Strain Rev. 1) Vaccine, Living
for Veterinary U se
Canine Adenovirus Vaccine (Inactivated) Canine Adenovirus Vaccine, Inactivated
Canine Adenovirus Vaccine (Live) Canine Adenovirus Vaccine, Living
Canine Distemper Vaccine (Live) Canine Distemper Vaccine, Living
Canine Parvovirosis Vaccine (Inactivated) Canine Parvovirus Vaccine, Inactivated
Canine Parvovirosis Vaccine (Live) Canine Parvo virus Vaccine, Living
Clostridium Botulinum Vaccine for Veterinary Use Clostridium Botulinum Vaccine
Botulinum Vaccine

Clostridium Chauvoei Vaccine for Veterinary Use Clostridium Chauvoei Vaccine
Blackleg Vaccine
Clostridium Novyi (Type B) Vaccine for Veterinary Use Clostridium Novyi Type B Vaccine
Black Disease Vaccine
Clostridium Novyi Alpha Antitoxin for Veterin~~ Use Clostridium Novyi Alpha Antitoxin
Clostridium Perfringens Beta Antitoxin for Veterinary Use Clostridium Perfringens Beta Antitoxin
Clostridium Perfringens Epsilon Antitoxin for Veterinary Use Clostridium Perfringens Epsilon Antitoxin
Clostridium Perfringens Type D Antitoxin
Clostridium Perfringens Vaccine for Veterinary Use Clostridium Perfringens Vaccines
Type B Clostridium Perfringens Type B Vaccine
Lamb Dysentery Vaccine
TypeC Clostridium Perfringens Type C Vaccine
Struck Vaccine
TypeD Clostridium Perfringens Type D Vaccine
Pulpy Kidney Vaccine
Clostridium Septicum Vaccine for Veterinary Use Clostridium Septicum Vaccine
Braxy Vaccine
Distemper Vaccine (Live) for Mustelids Ferret and Mink Distemper Vaccine, Living
Egg Drop Syndrome '76 Vaccine (Inactivated) Egg Drop Syndrome 76 (Adenovirus) Vaccine
Equine Herpesvirus Vaccine (Inactivated) Equine Herpesvirus Vaccine, Inactivated
Equine Influenza Vaccine (Inactivated) Equine Influenza Vaccine, Inactivated
Feline Calicivirosis Vaccine (lnactivated) Feline Calicivirus Vaccine, Inactivated
Feline Calicivirosis Vaccine (Live) Feline Calicivirus Vaccine, Living
Feline Infectious Enteritis (Feline Panleucopenia) Vaccine Feline Infectious Enteritis Vaccine, Inactivated
(Inactivated) Feline Panleucopenia Vaccine, Inactivated
Feline Infectious Enteritis (Feline Panleucopenia) Vaccine Feline Infectious Enteritis Vaccine, Living
(Live) Feline Panleucopenia Vaccine, Living
Feline Leukaemia Vaccine (Inactivated) Feline Leukaemia Vaccine, Inactivated
Feline Viral Rhinotracheitis Vaccine (Inactivated) Feline Viral Rhinotracheitis Vaccine, Inactivated
Feline Viral Rhinotracheitis Vaccine (Live) Feline Viral Rhinotracheitis Vaccine, Living
Foot-and-Mouth Disease (Ruminants) Vaccine (Inactivated) Foot and Mouth Disease (Ruminants) Vaccine
Fowl-pox Vaccine (Live) Fowl Pox Vaccine, Living
Furunculosis Vaccine (Inactivated, Oil-adjuvanted, Injectable) Furunculosis Vaccine for Salmonids, Inactivated
for Salmonids
Infectious Bovine Rhinotracheitis Vaccine (Live) Infectious Bovine Rhinotracheitis Vaccine, Living

Marek's Disease Vaccine (Live) Marek's Disease Vaccine, Living
Marek's Disease Vaccine (Turkey Herpes Virus)
Marek's Disease Vaccine, Living (HVT)
Neonaral Piglet Colibacillosis Vaccine (Inactivated) Porcine E. Coli Vaccine, Inactivated
Porcine Escherichia Coli Vaccine, Inactivated
Neonatal Ruminant Colibacillosis Vaccine (Inactivated) Ruminant E. Coli Vaccine, Inactivated
Ruminant Escherichia Coli Vaccine, Inactivated
Newcastle Disease Vaccine (Inactivated) Newcastle Disease Vaccine, Inactivated
Newcastle Disease Vaccine (Live) Newcastle Disease Vaccine, Living
Porcine Actinobacillosis Vaccine (Inactivated) Porcine Actinobacillosis Vaccine, Inactivated
Porcine Influenza Vaccine (Inactivated) Swine Influenza Vaccine, Inactivated
Porcine Parvovirosis Vaccine (Inactivated) Porcine Parvovirus Vaccine, Inactivated
Porcine Progressive Atrophic Rhinitis Vaccine (Inactivated) Porcine Progressive Atrophic Rhinitis Vaccine, Inactivated
Rabies Vaccine (Inactivated) for Veterinary Use Rabies Veterinary Vaccine, Inactivated
Rabies Vaccine (Live, Oral) for Poxes Rabies Vaccine for Poxes, Living
Swine Erysipelas Vaccine (Inactivated) Swine Erysipelas Vaccine, Inactivated
Tetanus Antitoxin for Veterinary Use Clostridium Tetani Antitoxin
Tetanus Antitoxin (Veterinary)
Tetanus Vaccine for Veterinary Use Clostridium Tetani Vaccines
Tetanus Toxoids (Veterinary)
(for vaccines with an appropriate potency)
Clostridium Tetani Vaccine for Equidae
Tetanus Toxoid for Equidae
Tuberculin Purified Protein Derivative, Avían Avían Tuberculin Purified Protein Derivative
Avían Tuberculin P .P.D .
Tuberculin Purified Protein Derivative, Bovine Bovine Tuberculin Purified Protein Derivative
Bovine Tuberculin P.P.D.
Vibriosis (Cold-water) Vaccine (Inactivated) for Salmonids Cold-water Vibriosis Vaccine for Salmonids, Inactivated
Vibriosis Vaccine (Inactivated) for Salmonids Vibriosis Vaccine for Salmonids, Inactivated
Yersiniosis Vaccine (Inactivated) for Salmonids Enteric Redmouth Disease Vaccine for Rainbow Trout,
Inactivated
Radiopharmaceutical Preparations
Pluorodopa e sp) Prepared by Electrophilic Substitution) Fluorodopa (I Sp) Injection
Injection
Technetium (99m Tc) Human Albumin Injection Technetium t 9m
Tc) Albumin Injection
V-AS90 Appendix XXI B 2014
When used in the practice of homoeopathy, certain substances that are the subject of a monograph in the European
Pharrnacopoeia have been known traditionally by a name other than that at the head of the Ph. Eur. monograph. The name or
names given in the right-hand column of the list below are Approved Synonyms that may be used only in relation to
homoeopathic preparations for the name at the head of a monograph in the European Pharrnacopoeia given in the left-hand
column.
EUROPEAN PHARMACOPOEIA TITLE APPROVED SYNONYM FOR HOMOEOPATHIC

USE
Substances used in Homoeopathy
Arnmonium Chloride Ammonium Muriaticum
Atropine Sulfate Atropinum Sulfuricum
Caraway Fruit Carum Carvi
Cascara Cascara Sagrada
Cholesterol Cholesterinum
Cinchona Bark China
Cinnamon Cinnamomum
Eucalyptus Leaf Eucalyptus
Bitter Fennel Foeniculum Vulgare
Lithium Carbonate Lithium Carbonicum
Magnesium Chloride Hexahydrate Magnesium Muriaticum
Magnesium Sulfate Magnesium Sulfuricum
Mercuric Chloride Mercurius Sublimatus Corrosivus
Mercurius Corrosivus
Potassium Chloride Kali Muriaticum
Quinine Sulfate Chininum Sulfuricum
Rhatany Root Ratanhia
Rhubarb Rheum Palmatum
Senega Root Senega
Silver Nitrate Argentum Nitricum
Sodium Carbonate Monohydrate Natrium Carbonicum
Sodium Chloride Natrium Muriaticum
Anhydrous Sodium Sulfate Natrium Sulfuricum
Valerian Root Valeriana
Zinc Sulfate Zincum Sulfuricum
2014 Appendix XXI e V-A591
C. Eye drops
Codes for eye drops in single-dose containers
The foIlowing codes are approved for use on single unit doses of eye drops where the individual container may be too small to
bear all of the appropriate labeIling information (se e the general monograph for Eye Drops) .
The inc1usion of a preparation in this list does not necessarily mean that a monograph is or will be inc1uded in the British
Pharmacopoeia.
Eye Drops Code Eye Drops Code
Adrenaline / Epinephrine ADN / EPN Metipranolol MPR

Aprac10nidine ACD Moxisylyte MOX
Atropine Sulfate ATR Neomycin NEO
Betamethasone BET Oxybuprocaine OXB
Carbachol CAR Phenylephrine PHNL
Castor Oil CASOIL Phenylephrine and Cyc1opentolate PHNCYC
Chloramphenicol CPL Pilocarpine PIL
Cocaine CCN Povidone-Iodine PVI
Cyc1opentolate CYC Prednisolone PRED
Dic10fenac DICL Proxymetacaine PROX
Fluorescein FLN Proxymetacaine and Fluorescein PROXFLN
Dexamethasone Sodium Phosphate DSP Rose Bengal ROS
Gentamicin GNT Sodium Chloride SALINE l
Homatropine HOM Sodium Cromoglicate SCG
Hydrocortisone HCOR Sulfacetamide SULF
Hydroxyethylcellulose and Sodium HECL Tetracaine TET
Chloride
Hyoscine HYO Timolol TIM
Hypromellose HPRM Tropicamide TRO
Lachesine LAC Zinc Sulfate ZSU
Lidocaine and Fluorescein LIDFLN
1 The term 'Saline' indicates the contents of the container are a 0.9% w/v solution of Sodium Chloride.
V-A592 Appendix XXII 2014
- the amount of material used to produce adose of

Appendix XXII medicinal product;
- controls carried out on the donor(s), on the raw material,
during production and on the final product;
A. Viral Safety
- the manufacturing process of the product and its capacity
(Ph Eur. general text 5.1 . 7) to remove andlor inactivate viruses.
This chapter provides general requirements conceming the
The risk assessment can be based mainly on the
viral safety of medicinal products whose manufacture has
manufacturing conditions if these include rigorous
involved the use of material s of human or animal origino
inactivation steps (for example, for gelatin etc., and products
Since viral safety is a complex issue, it is important that a risk
terminally sterilised by steam or dry heat as described in the
assessment is carried out. Requirements to be applied to a
general texts on sterility (5.1)).
specific medicinal product are decided by the competent
authority.
Where the risk of viral contamination exists, complementary
measures are used as appropriate to assure the viral safety of
medicinal products, based on:
B. Minimising the Risk of Transmitting
- selection of source materials and testing for viral Animal Spongiform Encephalopathy
contaminants; Agents Via Human and Veterinary
- testing the capacity of the production process to remove
ami/or inactivate viruses;
Medicinal Products
- testing for viral contamination at appropriate stages of (Ph. Eur. general text 5.2.8)
production. This chapter is identical with the Note for Guidance on
Minimising the Risk of Transmitting Animal Spongifoml
Where appropriate, one or more validated procedures for
Encephalopathy Agents via Human and Veterinary Medicinal
removal or inactivation of viruses are applied.
Products - Revision 3, (EMA /41O/0l rev. 3) .
Further detailed recommendations on viral safety, including
validation studies, are provided, in particular, by the Note for Contents
guidance on virus validatwn studies: the design, contribution and
l. INTRODUCTION
interpretatwn of studies validating the inactivation and removal of
viruses (CPMP/BWP/268/95) of the Committee for Proprietary 1-l. Scientific background
Medicinal Products, and the ICH guideline Q5A: Viral safety 1-2. Regulatory compliance
evaluation of biotechnology products derived from celllines of 2. SCOPE
human or animal origin (including any subsequent revisions of 3. GENERAL CONSIDERATIONS
these documents) .
3-l. Scientific principIes for minirnising risk
Requirements conceming immunological products for
3-2. Animal source
veterinary use are dealt with in the monographs Vaccines for
veterinary use (0062) and Immunosera for veterinary use (0030) 3-2-l. Geographical sourcing
and related general chapters. 3-2-1-l. Bovine materials
3-2-1-2. Sheep and goats (small ruminants)
Ftisk assessnaent
3-2-2. BSE negligible risk (closed) bovine herds
A risk assessment with respect to viral safety is carried out
3-3. Animal parts, body fluids and secretions as starting
where materials of human or animal origin are used as
material
ingredients of medicinal products or in the manufacture of
active substances, excipients or medicinal products. 3-4. Age of animals
The principIe of the risk assessment is to consider various 3-5. Manufacturing Process
factors that may influence the potential level of infectious 4. RISK ASSESSMENT OF MATERlALS OR
particles in the medicinal product and factors related to the SUBSTANCES USED IN THE
use of the medicinal product that determine or influence the MANUFACTURE AND PREPARATION OF A
viral risk to the reoipients. MEDICINAL PRODUCT IN THE CONTEXT
The risk assessment takes into consideration relevant factors, OF REGULATORY COMPLIANCE
for example: 5. BENEFIT/RISK EVALUATION
- the species of originó 6. SPECIFIC CONSIDERATIONS
- the organ, tissue, fluid of originó 6-l. Collagen
- the potential contaminants in view ()f the origin of the raw 6-2. Gelatin
material and the history of the donor(s), preferably
6-3. Bovine blood and blood derivatives
including epidemiological data;
6-4. Tallow derivatives
- the potential contaminants from the manufacturing
process (for example, from risk material s used during 6-5. Animal charco al
manufacture); 6-6. Milk and milk derivatives
- the infectivity and pathogerucity of the potential 6-7. Wool derivatives
contaminants for the intended recipients of the medicinal 6-8 . Amino acids
product, taking account of the route of administration of 6-9. Peptones
the medicinal product;
2014 Appendix XXII B V-A593
1. Introduction few classical BSE cases have been found like Canada or
1-1 Scientific background USA. The atypical BSE agent has been experimentally
Transmissible Spongiform Encephalopathies (TSEs) are transmitted to transgenic mice expressing the human prion
chronic degenerative nervous diseases characterised by the protein and to a cynomolgus monkey.
accumulation of an abnormal isoform of a cellular Scrapie occurs worldwide and has been reported in most
glycoprotein (known as PrP or prion protein). The abnormal European countries. It has the highest incidence in Cyprus.
isoform of PrP (PrpTSE) differs from normal PrP (PrPc) in While humans have been exposed to naturally occurring
being highly resistant to protease and heat denaturation scrapie for over 250 years, there is no epidemiological
treatments. PrpTSE is considered to be the infective agent evidence directly linking scrapie to spongiform
responsible for transmitting TSE disease. encephalopathies in humans 1 . However, there remains a
TSE diseases in animals include: theoretical and currently unquantifiable risk that sorne
- bovine spongiform encephalopathy (BSE) in cattle, BSE-contaminated protein supplement may have been fed
- scrapie in sheep and goats, to sheep. Further, it should also be assumed that any BSE
agent introduced into the small ruminant population via
- chronic wasting disease (CWD) in cervids (deer and elk),
contaminated feed is likely to be recycled and amplified2 .
- transmissible mink encephalopathy (TME) in farmed
mink, There is interest in infecting cells with TSE agents to develop
assays and for basic scientific reasons. Sorne success has been
- feline spongiform encephalopathy (FSE) in felids
reported, usually but not always with neural celllines.
(specifically domestic cats and captive large cats), and
The conditions needed to infect a cell are not well
- spongiform encephalopathy of exotic ungulates in zoos.
understood and the process is difficult requiring particular
In humans, spongiform encephalopathies include different combinations of agent and cell. It is not considered
forms of Creutzfeldt-Jakob Disease (CJD), Kuru, appropriate to make specific recommendations in terms of
Gerstmann-Straussler-Scheinker Syndrome (GSS), and Fatal cell substrates to be used for production of
Familial Insomnia (FFI). biological/biotechnology-derived substances. Nevertheless, the
Iatrogenic transmission of spongiform encephalopathies has possibility of infection of celllines with TSE agents should be
been reported. In sheep, scrapie has been accidentally taken into account in risk assessments.
transmitted by the use of Louping III vaccine prepared from
pooled, formaldehyde treated ovine brain and spleen in 1-2 Regulatory compliance
which material from scrapie-infected sheep had been Risk assessment Since the use of animal-derived materials
inadvertently incorporated. Also, transmission of scrapie is unavoidable for the production of sorne medicinal
to sheep and goats occurred following use of a products and that complete elimination of risk at source is
formol-inactivated vaccine against contagious agalactia, rarely possible, the mea sures taken to manage the risk of
prepared with brain and mammary gland homogenates transmitting animal TSEs via medicinal products represent
of sheep infected with Mycoplasma agalactiae. In man, risk minimisation rather than risk elimination. Consequently,
cases of transmission of CJD have been reported which have the basis for regulatory compliance should be based on a risk
been attributed to the parenteral administration of growth assessment, taking into consideration all pertinent factors as
hormone and gonadotropin derived from human cadaveric identified in this chapter (se e below) .
pituitary glands. Cases of CJD have also been attributed to Legal basis The note for guidance is published by the
the use of contaminated instruments in brain surgery and European Commission following
with the transplantation of human dura mater and cornea. - Annex I, part I, module 3, section 3.2: Content: basic
Interspecies TSE transmission is restricted by a number of principies and requirements, point (9) of Directive
natural barriers, transmissibility being affected by the species 2001/83/EC of the European Parliament and of the
of origin, the prion strain, dose, route of exposure and, in Council of 6 November 2001 on the Community code
sorne species, the host allele of the PRNP gene. Species relating to medicinal products for human use 3, as
barriers can be crossed under appropriate conditions. amended, and
BSE was first diagnosed in the United Kingdom in 1986 and - Annex I, Title 1, part 2, section C Production and control 01
a large number of c'attle and individual herds have been stal1ing material of Directive 2001/82/EC of the European
affected. It is clear that BSE is a food borne disease Parliament and of the Council of 6 November 2001 on
associated with feed (e.g. meat and bone meal) derived from the Community code relating to veterinary medicinal
TSE affected animals. Other countries have experienced products 4, as amended.
cases of BSE, either in animals imported from the United These directives require that applicants for marketing
Kingdom or in indigenous animals. There is convincing authorisation for human and veterinary medicinal products
evidence to show that the variant form of CJD (vCJD) is must demonstrate that medicinal products are manufactured
caused by the agent which is responsible for BSE in cattle. in accordance with the latest version of this note for guidance
Therefore, a cautious approach continues to be warranted if published in the Official Journal 01 the European Uníon . This is
biological materials from species naturally affected by TSE a continuing obligation after the marketing authorisation has
diseases, especially bovine species, are used for the been granted.
manufacture of medicinal products.
In the course of active surveillance programs, two previously J This is eUn"emly being assessed by EFSA and ECDC. For updated
unrecognized forms of atypical BSE (BSE-L, also named information, please refer 10 thefollowing link :hup:llregisterofquestions.efsa
BASE, and BSE-H) have been identified in rare sporadic . europa. el.llroqFrontendlql.lestionsListLoader?mandate=M -2009-0221
2 In Janl.lary 2005, afier confirmation of BSE in a goat in Franee,
cases from Europe, North America, and Japan. The 'L' and
additionallegislarive meaSl.lres were taken related 10 monitoring and an
'H' identify the higher and lower electrophoretic positions of increased testing of small ruminanrs. The inereased sl.lrveillanee did noc
their protease-resistant PrpTSE isoforms. It is noteworthy that idemify addirional cases of BSE in sheep and goars in the EU.
atypical cases have been found in countries that did not J OJ L 311,28.11.2001, p. 67.
experience classical BSE so far, like Sweden, or in which only 4 OJ L 311,28. 11.2001, p. 1.
V-A594 Appendix XXII B 2014
By definition, the principIe of Specified Risk Materials as Materials

defined in Regulation (EC) No 999/2001 ofthe European This chapter is concerned with materials derived from
Parliament and of the Council 5 does not apply to medicinal "TSE-relevant animal species" that are used for the
products. However, Regulation (EC) No 1774/2002 of the preparation of :
European Parliament and of the Council6, which applies - active substances,
since 1st May 2003, lays down health rules conceming
- excipients and adjuvants, and
animal by-products not intended for human consumption.
As a general rule, and unless properly justified, all animal - raw and starting materials and reagents used in
by-products used as starting materials in the manufacture of production (e.g. bovine serum albumin, enzymes, culmre
medicinal products should be ' Category 3 (i.e. safe) materials media inc1uding those used to prepare working cell banks,
or equivalent', as defined in Regulation (EC) No 1774/2002. or new master cell banks for medicinal products which are
Justification for the use of substances derived from other, subject to a new marketing authorisation) .
high infectivity materials must follow an appropriate This chapter is also applicable to materials that come into
benefit/risk evaluation (see further below). direct contact with the equipment used in manufacmre of the
The note for guidance should be read in conjunction with medicinal product or that come in contact with the medicinal
the various EU legal instruments inc1uding Commission product and therefore have the potential for contamination.
decisions progressively implemented since 1991 . Where Materials used in the qualification of plant and equipment,
appropriate, references to these decisions are given in the such as culture media used in media fill experiments to
texto Position statements and explanatory notes made by the validate the aseptic filling process, shall be considered in
Committee for Medicinal Products for Human Use (CHMP) compliance with this chapter provided that the constiment or
and Committee for Medicinal Products for Veterinary Use constiments are derived from tissues with no detectable
(CVMP) are still applicable for the purpose of regulatory infectivity (category lC tissues), where the risk of cross-
compliance unless otherwise superseded by the note for contamination with potentially infective tissues has been
guidance. considered (see section 3-3) and where the materials are
The general monograph Products with risk of transmitting sourced from countries with negligible BSE risk or controlled
agents of animal spongifoml encephalopathies of the European BSE risk (Categories A and B, respectively - see section 3-2).
Pharmacopoeia refers to this chapter, which is identical with Such information shall be provided in the dossier for a
the note for guidance. The monograph forms the basis for marketing authorisation and verified during routine
issuing Certifica tes of Suitability as a procedure for inspection for compliance with Good Manufacturing Practice
demonstrating TSE compliance for substances and materials (GMP).
used in the manufacture of human and veterinary medicinal Other material s such as c1eaning agents, softeners and
products. lubricants that come into contact with the medicinal product
Clarification of note for guidance As the scientific during its routine manufacture or in the finishing stage or in
understanding of TSEs, especially the pathogenesis of the the primary packaging are considered in compliance with this
diseases, is evolving, from time to time CHMP and its chapter if they are tallow derivatives prepared using the
Biologics Working Party in collaboration with CVMP and its rigorous physicochemical processes as described in section 6.
lmmunologicals Working Party may be required in the future SEED LOTS, CELL BANKS AND ROUTINE
to develop supplementary guidance in the form of position FERMENTATIONIPRODUCTIO~
statements or explanatory notes for the purpose of c1arifYing For the purpose of regulatory compliance, master seeds or
the note for guidance. The supplementary guidance shall be master cell banks in marketing authorisation applications
published by the Commission and on the website of the lodged after 1 July 2000 (for human medicinal products) or 1
European Medicines Agency and taken into consideration October 2000 (for veterinary medicinal products) shall be
accordingly in the scope of the certification of the European covered by the note for guidance.
Directorate for the Quality of Medicines & HealthCare
(EDQM). Master seeds and master cell banks,
- for vaccine antigens,
2. Scope - for a biotechnology-derived medicinal product as
TSE-relevant animal species described in the Annex to Regulation (EC) No 726/2004
Cattle, sheep, goats and animals that are naturally susceptible of the European Parliament and of the Counc;iI 10, and
to infection with transmissible spongiform encephalopathy - for other medicinal products using seed lots or cell
agents or susceptible to infection through the oral route other banking systems in their manufacture,
than humans 7 and non-human primates are defined as that have already been approved for the manufacture of a
"TSE-relevant animal species"s. constiment of an authorised medicinal product shalJ be
considered in compliance with the note for guidance even if
5 OJ L 147, 31.5.2001, p. 1. they are incorporated in marketing authorisation applications
6 OJ L 273,10.10.2002, p. 1. Regulation (EC) 1774/2002 has been lodged after 1 July 2000 (for human medicinal products) or 1
repealed by R egulation (EC) 1069/2009 that wil/ apply from 4 March 2011 October 2000 (for veterinary medicinal products).
(OJ L 300, 14.11.2009, p. 1).
7 Regulatory guidance and positian papers have been issued by the
Committee fo1' Medicinal PrOdUClS for Human Use and its Biologics Wo1'king
Party on human tissue derived medicinal products in relation to CJD and
vCJD. Such guidance can be found on hnp: //www. ema. europa.eu 9 See a/so: Position papel' on the assessmellt of the 11sk of transmission of
8 Pigs and birds, which are animal species of particular interest for the animal spongiform encephalopathy agents by master seed materia/s used in
production of medicinal products, are not naturally susceptible 10 infection via the production ofveterinalY vaccines (EMEA /CVMP/019/01-February 2001
the oral l'Oute. Therefore they are not TSE-relevant animal species within the adopted by ¡he Com11úttee for Medicinal Products for VeterinalY Use
meaning of this chapter. A/sa dogs, rabbits and fish are non TSE-relevant (CVMP) inJuly 2001, (OJ C 286,12. 10.2001, p. 12)).
animal species within the meaning of this chapter. 10 OJ L 136, 30.4.2004, p. 1.
2014 Appendíx XXII B V-A595
Master celI banks and master seeds established before 1 July - production process(es) including the quality assurance
2000 (for human medicinal products) or 1 October 2000 (for system in place to ensure product consistency and
veterinary medicinal products), but not yet approved as a traceability .
constiruent of an authorised medicinal product shall 3-2 Animal source
demonstrate that they fulfil the requirements of the note for
The source materials used for the production of materials for
guidance. If, for sorne raw or starting materials or reagents
the manufacrure of medicinal products shalI be derived from
used for the establishment of these cell banks or seeds, fulI
animals fit for human consumption following ante- and
documentary evidence is no longer available, the applicant
post-mortem inspection in accordance with EU or equivalent
should present a risk assessment as described in Section 4 of
(third country) conditions, except for materials derived
the note for guidance.
from live animals, which should be found healthy after
Established working seeds or celI banks used in the clinical examination.
manufacrure of medicinal products authorised before 1 July
3-2-1 GEOGRAPHICAL SOURCING
2000 (human medicines) or 1 October 2000 (veterinary
3-2-1-1 Bovine materials
medicines), which have been subjected to a properly
The World Organisation for Animal Health (OlE)ll lays
conducted risk assessment by a Competent Authority of the
Member Sta tes or the European Medicines Agency and down the criteria for the assessment of the starus of countries
declared to be acceptable, shall also be considered compliant. in the chapter of the Intemational Animal Health Code on
bovine spongiform encephalopathy. Countries or regions are
However, where materials derived from the "TSE-relevant classified as folIows :
animal species" are used in fermentation/routine production
A. countries or regions with a negligible BSE risk;
processes or in the establishment of working seeds and
working celI banks, ·the applicant must demonstrate that they B. countries or regions with a controlIed BSE risk ;
fulfil the requirements of the note for guidance. C. countries or regions with an undetermined BSE risk.
As stipulated in Commission Regulation (EC) No 999/2001,
3. General Considerations as amended 12, the classification of countries or regions
3-1 Scientific principIes for minimising risk thereof according to their BSE risk, based on the rules laid
When rnanufacturers have a choice, the use of materials from down by OlE, is legalIy binding in the EU since 1 July 2007.
"non TSE-relevant animal species" or non-animal origin is Commission Decision 2007/453/EC 13 as amended, provides
preferred. The rationale for using rnaterials derived from the classification of countries or regions according to their
"TSE-relevant animal species" instead of materials from BSE risk.
"non-TSE-relevant species" or of non-animal origin should Previously, the European Commission Scientific Steering
be given. If materials from "TSE-relevant animal species" Committee (SSC) 14 had established a temporary system for
have to be used, consideration should be given to alI the classifying the countries according to their geographical BSE
necessary measures to minimise the risk of transmission of risk (GBR) 15.
TSE. For the purposes of this chapter the BSE classification based
Readily applicable diagnostic tests for TSE infectivity in vivo on the OIE rules should be used. If a country, which was
are not yet available. Diagnosis is based on post-mortem previously classified in accordance to the SSC GBR criteria,
confirmation of characteristic brain lesions by histopathology has not been classified yet according to the OIE rules, the
and/or detection of PrpTSE by Westem blot or irnmunoassay. GBR classification can be used until OIE classification has
The demonstration of infectivity by the inoculation of suspect taken place, provided that there is no evidence of significant
tissue into target species or laboratory animals is also used for change in its BSE risk16 .
confirmation. However, due to the long incubation periods of Where there is a choice, animals should be sourced from
alI TSEs, results of in vivo tests are available on1y after countries with the lowest possible BSE risk (negligible BSE
months or years. risk countries (Category A)) unless the use of material from
Several immunochemical tests have been developed for the countries with a higher BSE risk is justified. Sorne of the
detection of PrpTSE in post-mortem samples and sorne are 11 hrrp: //www.oie.int/eng/Smrus/BSElen_BSE-free.htm
now considered to be extremely sensitive. However, their 12 Regulation (EC) No 722/2007 (OJ L 164, 26.6.2007, p. 7)
ability to detect an infected animal depends on the timing of 13 OJ L 172, 30.6.2007, p. 84
sample collection in relation to timing of exposure, the type 14 The Seientifie Steenng Committee established by Commission Decision
of tissue collected and infectious dose acquired, together with 97/404/EC (OJ L 169, 27.6.1997, p. 85) shall assist the Commission to
consequential timing of onset of clinical disease. There is obtain the besr scientifie advice available on matters relating to eonsumer
currently insufficient information on how this might be health Sinee May 2003, its tasks have been mken over by the European
affected by strain variations. Food Safety Authonty (EFSA): http://www.efsa.europa.eu
15 The European Scientifie Steering Committee classifieation for geographieal
Although screening of source animals by in vitro tests may BSE risk (GBR) gives an indication of tite leve! of likelthood of the presence
prevent the use of animal s at late stages of incubation of the of one or more cattle clinieally or pre-clinieally infecred with BSE in a given
disease and may provide information about the eountry or region. A definition of the four eategones is provided in the
epidemiological status of a given country or region, none of following Table.
the tests are considered suitable to unambiguously confirm GBR ¡evel Preu nte of Que 01 more cattle cJinlcally Of pre-cJlnica1ly Infccted wlth BSE In a geographical
the negative starus of an animal. Minimising the risks of re 'oncounlr
IIighlyunlikeJ}'
transmission of TSE is based upon three complementary JI Unli kely butnotexcl ude d
parameters: DI Like!y bul not confirmed or conDrmed al a lower level

IV Confirmed at a highcr level (~ 100 cascs!] i\lillio n ad ult catt lc per ycar)
- the source animals and their geographical origin,
Reports of the GBR assessment of the eountries are available on the SSC
- nature of animal material used in manufacture and any website (http: //ee.europa.eulfoodlfs/sc/ssc/outeome_en.html)
procedures in place to avoid cross-contamination with 16 Experts consider that the GBR classification system is smble enough, so
higher risk material s, that it ean eontinue ro be used, dunng the intenm period, for the
demonstration of eompliance with this ehapter.
materials identified in Section 6, "Specific Conditions" can summarise current data about the distribution of infectivity
be sourced from countries with controlled BSE risk and Prp T SE in cattle with BSE, and in sheep and goats with
(Category B) and, in sorne cases, from countries with scrapie 20 .
undetermined BSE risk (Category C), provided that the The information in the tables is based exclusively upon
controls and requirements as specified in the relevant sections observations of naturally occurring disease or primary
below are applied. Apart from these exceptions, animals must experimental infection by the oral route (in cattle) but does
not be sourced from countries with undetermined BSE risk not include data on models using strains of TSE that have
(Category C), and justifications for the use of animals from been adapted to experimental animals, because passaged
countries with undetermined BSE risk (Category C) must strain phenotypes can differ significantly and unpredictably
always be provided. from those of naturally occurring disease. Because
3-2-1-2. Sheep and goats (small ruminants) irnmunohistochemical andlor Western blot detection of
Naturally occurring clinical scrapie cases have been reported misfolded host protein (PrpTSE ) have proven to be a
in a number of countries worldwide. As BSE in sheep and surrogate marker of infectivity, Prp TSE testing results have
goats could possibly be mistaken for scrapie, as a been presented in parallel with bioassay data. Tissues are
precautionary measure, sourcing of materials derived from grouped into three major infectivity categories, irrespective of
small ruminants shall take into account the prevalence of the stage of disease:
both BSE and scrapie in the country and the tissues from
which the materials are derived. Category lA High·in(ectivity tissues
centralnervous system (CNS) tissues that attain a high titre of infectivíty in the latcr
The principies related to "BSE negligible risk (closed) bovine slaltes of all TSEs and certain tiMues that are anatomicallv associated with the CNS
Category lB Lower-infectivity tissues
herds" (see section 3-2-2) could equally be applied in the
peripheral tissues that have tested positive ror infectivity and/ or PrpTSt in at lcast onc
context of srnall ruminants in order to develop a framework form o(TSE
to define the TSE status of a fiock of small ruminants. Category le Tissues with no detectable infectivily
tissues that ha\'e been examincd for infecti\'ity, without <In)' infee!;v;t)' detected, and/or
Por sheep, because of the concern over the possibility of BSE PrprsE with negath,c results
in sheep, the use of a genotype(s) showing resistance to
BSE/scrapie infection could be considered in establishing Category lA tissues and substances derived from them shall
TSE free fiocks 17 . However, the possibility that genotypes not be used in the manufacture of medicinal products, unless
resistant to scrapie could be susceptible to BSE (experimental justified (see Section 5).
oral exposure) or atypical scrapie (natural cases) should also
be taken into account. Goats have not been studied Although the category of lower risk tissues (category lB
tissues) almost certainly ineludes sorne (e.g. blood) with a
sufficiently with regard to a genotype specific sensitivity.
lower risk than others (e.g. Iymphoreticular tissues), the data
Material of small ruminant origin should preferably be about infectivity levels in these tissues are too limited to
sourced from countries with a long history of absence of subdivide the category into different levels of risk. lt is also
scrapie. Justification shall be required if the material is evident that the placement of a given tissue in one or another
sourced from sorne other origino category can be disease and species specific, and subject to
3-2-2. BSE negligible risk (c1osed) bovine herds The revision as new data emerge.
safest sourcing is from countries or regions with a negligible
Por the risk assessment (se e section 4), manufacturers andlor
risk (Category A countries). Other countries may have or
marketing authorisation holders/applicants shall take into
have had cases of BSE at sorne point in time and the
account the tissue elassification tables in the Annex to this
practical concept of "Negligible risk (closed) bovine herds"
chapter.
has been developed by the SSC and endorsed by the CHMP
and CVMP. Criteria for establishing and maintaining a The categories in the tables are only indicative and it is
"BSE negligible risk (closed) bovine herd" can be found in important to note the following points.
the SSC opinion of 22-23 July 1999 18 . - In certain situations there could be cross-contarnination
Por the time being it is not possible to quantify the reduction of tissues of different categories of infectivity.
of the geographical BSE risk for canle from BSE 'negligible The poten ti al risk will be influenced by the circumstances
risk (closed) bovine herds'. However, it is expected that this in which tissues were removed, especially by contact of
risk reduction is substantial. Therefore, sourcing from such tissues with lower-infectivity tissues or no detectable
closed bovine herds shall be considered in the risk assessment infectivity (categories lB and IC tissues) with
in conjunction with the OIE classification of the country. high-infectivity tissues (category lA tissues). Thus,
cross-contarnination of sorne tissues may be increased if
3-3 Animal parts, body fluids and secretions as
starting materials infected animals are slaughtered by brail). stunning
(penetrative or non penetrative) or if the brain andlor
In a TSE infected animal, different organs and secretions spinal cord is sawed. The risk of cross-contamination will
have different levels of infectivity. lf materials from be decreased if body fiuids are collected with minimal
'TSE-relevant animal species' have to be used, consideration damage to tissue and cellular components are removed,
should be given to use materials of the lowest category of and if foetal blood is collected without contamination
risk. The tables given in the Annex of this chapter 19 from other maternal or foetal tissues including placenta,
17 Opinion of the Scientific Panel on Biological Hazards on 'the breeding amniotic and allantoic fluids. Por certain tissues, it is very
programme fo1' TSE resisrance in sheep ':hup: Ilwww.efsa.europa.euIEFSA I difficult or impossible to prevent cross-contamination with
efsa_locale-1178620753812_1178620775678. htm category lA tissues (e.g. skull). This has to be considered
/ 8 SSC Scientific Opinion on the conditions 1'elated to "BSE Negligible Risk in the risk assessment.
(Closed) Bovine Herds " adopted at the meeting of 22-23 July 1999.
hup: 1lec. europa.eu/food/fslsc/ssc/out56_en. html
/9 The tissue classification tables are based upon the most recent WHO 20 A Sciemific opinion on BSEITSE infectiviry in s111all nl1ninam tissues is
Guidelines on Tissue Infectiviry Distribution in Transmissible Spongifonn currently being reviewed by EFSA (Question No EFSA-Q-2010-052).
Encephalopathies (2010) For updated informatiol1 please follo w this link:http://1'egisterofqu estions.efsa
hup:1Iwww.who.imlbloddproductsltables tissueinfectiviry. pdf . europa. eulroq FromendlquestionsListLoader?mandate=M -2010-0041
- For certain classes of substances the 3-5. MANUFACTURING PROCESS

stunning/slaughtering techniques used may be The assessment of the overaJl TSE risk reduction of a
important in detertnining the potential risk21 because of medicinal product shaJl take into account the control
the likelihood of disseminating the brain particJes into the measures instituted with respect to :
peripheral organs, particularly to the lungs. - sourcing of the raw/starting materials, and
Stunning/slaughtering techniques should be described as
well as the procedures to remove high infectivity tissues. - the manufacturing process.
The procedures to collect the animal tissues/organs to be Controlled sourcing is a very important criterion in achieving
used and the measures in place to avoid cross- acceptable safety of the product, due to the documented
contamination with a higher risk material must also be resistance of TSE agents to most inactivation procedures .
described in detail. A quality assurance system, such as ISO 9000 certification,
- The risk of contamination of tissues and organs with HACCp 22 or GMP, must be put in place for monitoring the
BSE-infectivity potentially harboured in central nervous production process and for batch delineation (i.e.definition of
material as a consequence of the stunning method used batch, separation of batches, cleaning between batches).
Procedures shall be put in place to ensure traceability as weJl
for cattle slaughtering depends on the following factors :
as self-auditing and to auditing suppliers of raw/starting
- the amount of BSE-infectivity in the brain of the materials.
slaughtered animal,
Certain production procedures may contribute considerably
- the extent of brain damage, to the reduction of the risk of TSE contamination,
- the dissemination of brain particJes in the animal body. e.g. procedures used in the manufacture of tallow derivatives
These factors must be considered in conjunction with (see section 6). As such rigorous processing cannot be
the OIE/GBR cJassification of the source animals, the applied to many products, processes involving physical
age of the animals in the case of cattle and the post- removal, such as precipitation and filtration to remove prion-
mortem testing of the canle using a validated method. rich material, are likely to be more appropriate than chemical
The underlying principIes indicated aboye would be treatments. A description of the manufacturing process,
equally applicable to sheep and goats. including in-process controls applied, shaJl be presented and
The risk posed by cross-contamination will be dependent on the steps that might contribute to reduction or elimination of
TSE contamination should be discussed. Whenever different
several complementary factors including:
manufacturing sites are involved, the steps perfortned at each
- measures adopted to avoid contamination during site shall be clearly identified. The measures in place in order
collection of tissues (see aboye), to ensure traceability of every production batch to the source
- level of contamination (amount of the contaminating material should be described.
tissue), Cleaning process Cleaning of process equipment may be
- amount and type of materials collected at the same time. difficult to validate for the elimination of TSE agents. It is
Manufacturers or the marketing authorisation reported that after exposure to high titre preparations of TSE
holders/applicants should take into account the risk with agent, detectable infectivity can remain bound to the surface
respect to cross-contamination. of stainless steel. The removal of all adsorbed protein by the
use of 1 M sodium hydroxide or chlorine releasing
3-4 Age 01 animals
disinfectants (e.g. 20 000 ppm chlorine for 1 h) have been
As the TSE infectivity accumulates in bovine animals over an considered acceptable approaches where equipment that
incubation period of several years, it is prudent to source cannot be replaced has been exposed to potentially
from young animals. contaminated material. Milder treatments with limited
Presence of infectious material has essentially been reported concentrations of alkali or stabilized bleach, when properly
in the central nervous system and related tissues, as well as in fortnulated with detergents and used at specified
the Iymphoreticular system, depending on the TSE agent temperatures, have been shown to exhibit similar efficiency
(BSE in canle or scrapie in sheep and goat). The exact time for removing prions as did classical NaOH or chlorine
course of infectivity in the respective body parts and tissues, treatments. A system based on vaporised hydrogen peroxide
from the date of infection, is not known in both species and, also appeared to be efficient for inactivating TSE agents.
as such, it is difficult to give clear guidance on the age aboye These new treatments are more compatible with delicate
which the various tissues may be infected and should not be materials and may be suitable for practical use 23 .
colJected. The initial recommendation to collect tissues in the If risk materials are used in the manufacture of a product,
youngest age is stiJl valido In addition, it is noteworthy that cleaning procedures, including control mea sures, shaJl be put
the age criteria depend also on the geographical origino Age is in place in order to minimise the risk of cross-contamination
a more important parameter for materials from countries between production batches . This is especiaJly important if
where the risk is higher (Category B and C countries), than materials from different risk categories are handled in the
from countries with a negligible BSE risk (Category A same plant with the same equipment. In the case of using
countries). category lA materials in the manufacture of a product,
dedicated equipment shall be used, unless otherwise justified.
Further research is needed to develop and validate new
21 SSC opinion on stunning methods and BSE risk decontamination procedures to lower the risk of
(The risk oj dissemination oj brain panicles into the blood and carcass cross-contamination for material and devices which are not
when applying cerrain stunning methods), adopted at the meeting oj compatible with WHO-recommended procedures .
10-11 January 2002. http://ec.europa.eu/joodljs/sc/ssclout245_en.pdj. Report
oj the EFSA Working group on BSE risk from disseminatwn oj brain 22Hazard Analysis Critical Control Point.
particles in blood and carcass. Question No EFSA-Q-2003-122, adopted on 23WHO Guidelines on Tissue Injectiviry Distributwn in Transmissible
21 October 2004,http://www.ejsa.europa.eu/EFSA / Spongijorm Encephalopathies (2006) http://www.who.int/bloodproducts/tse/
ejsa_locale-1178620753812_1178620777397.htm WHO%20TSE%20Guidelines%20FINAL-22%20JuneupdatedNL.pdj
RemovallInactivation validation Validation studies of 5. BenefitlRisk Evaluation

removal/inactivation procedures for TSEs can be difficult to In addition to the parameters as mentioned in sections 3
interpreto It is necessary to take into consideration the nature (that may be covered by a TSE Certificate of Suitability
of the spiked material and its relevance to the natural issued by the EDQM) and 4, the acceptability of a particular
situation, the design of the study (inc1uding scaling-down of medicinal product containing materials derived from a
processes) and the method of detection of the agent (in vitro "TSE-relevant animal species", or which as a resuIt of
or in vivo assay) . Further research is needed to develop an manufacture could contain these materials, shaIl take into
understanding of the most appropriate "spike preparation" account the foIlowing factors :
for validation studies. Therefore, validation studies are
- route of administration of the medicinal product,
currently not generally required. However, if c1aims are made
for the safety of the product with respect to TSEs based on - quantity of animal material used in the medicinal product,
the ability of manufacturing processes to remove or inactivate - maximum therapeutic dosage (daily dose and duration of
TSE agents, they must be substantiated by appropriate treatment),
investigational studies 24 . - intended use of the medicinal product and its clinical
In addition to appropriate sourcing, manufacturers are benefit,
encouraged to continue their investigations into removal and - presence of a species barrier.
inactivation methods to identify steps/processes that would High-infectivity tissues (category lA tissues) and substances
have benefit in assuring the removal or inactivation of TSE derived thereof shaIl not be used in manufacture of medicinal
agents. In any event, a production process wherever possible products, their starting materials and intermediate products
shall be designed taking account of available information on (inc1uding active substances, excipients and reagents), unless
methods which are thought ro inactivate or remove TSE justified. A justification why no other materials can be used
agents. shaIl be provided. In these exceptional and justified
For certain types of products (see section 6-3 Bovine blood circumstances, the use of high-infectivity tissues could be
and blood derivatives), where validated removal/inactivation envisaged for the manufacture of active substances, when,
is not readily applicable, process evaluation might be after performing the risk assessment as described in Section 4
required. This should be based on the starting material and of this chapter, and taking into account the intended c1inical
any published data on TSE risk. use, a positive benefit/risk assessment can be presented by
the marketing authorisation applicant. Substances from
4. Risk Assessment of Materials or Substances Used category IA material s, if their use is justified, must be
in the Manufacture and Preparation of a Medicinal produced from animals of countries with negligible BSE risk
Product in the Context of Regulatory Compliance (Category A) .
The assessment of the risk associated with TSE needs careful
consideration of all of the parameters as outlined in section 6. Specific Considerations
3-1 (Scientific PrincipIes for Minimising Risk). The foIlowing material s prepared from "TSE-relevant animal
As indicated in the introduction to this chapter, regulatory species" are considered in compliance with this chapter
compliance is based on a favourable outcome from a risk provided that they meet at least the conditions specified
assessment. The risk assessments, conducted by the below. The relevant information or a certificate of suitability
manufacturers andlor the marketing authorisation holders or granted by the EDQM shaIl be provided by the marketing
applicants for the different materials or substances from authorisation applicant/holder.
" TSE-relevant animal species" used in the manufacture of a 6-1 Collagen
medicinal product shalI show that all TSE risk factors have
CoIlagen is a fibrous protein component of mammalian
been taken into account and, where possible, risk has been
connective tissue. For colIagen, documentation to
minimised by application of the principIes described in this
demonstrate compliance with this chapter needs to be
chapter. TSE Certifica tes of suitability issued by the EDQM
provided taking into account the provisions Iisted in sections
may be used by the marketing authorisation holders or
3 to 5. In addition, consideration should be given to the
applicants as the basis of the risk assessments.
following.
An overalI risk assessment for the medicinal product,
- For coIlagen produced from bones, the conditions
conducted by the marketing authorisation holders or
specified for gelatin are applicable (see below). Lower
applicants, shaIl take into account the risk assessments for aIl
inactivation capacity is expected from the coIlagen
the different materials from " TSE-relevant animal species"
manufacturing process than from that of gelatin.
and, where appropriate, TSE reduction or inactivation by the
Therefore, sourcing becomes a more critical aspect to
manufacturing steps of the active substance and/or finished
considero
producto
- CoIlagen produced from tissues such as hides, skins,
The final determination of regulatory compliance rests with
tendons and sinews do not usuaIly present a mea su rabIe
the competent authority. Ir is incumbent upon the
TSE risk provided that contamination with potentiaIly
manufacturers andlor the marketing authorisation holders or
infected materials, for example spilIage of blood andlor
applicants for both human and veterinary medicinal products
central nervous tissues, is avoided during procurement.
to select and justify the control measures for a given
Therefore, hides represent a safer raw material for human
"TSE-relevant animal species" derivative, taking into account
implants derived from coIlagen. However, cross-
the latest scientific and technical progress.
contamination with brain material released during the
slaughtering process that may have dried on the surface of
hides would be difficuIt to eliminate. This is another
aspect to consider in the evaluation of safety of this source
24 Guideline on the investigation of manufacturing process for plasma-derived
material.
medicinal products with regard 10 vCJD risk CPMPIBWPI5136103
The collagen manufacturing process can have sorne steps in provided that control measures are put in place to avoid
common with the manufacture of gelatin such as alkaline and cross-contamination both during the procurement of the
sodium sulphate treatment, calcium hydroxide and sodium hides and during the manufacturing process.
hydroxide treatments or enzyme treatment. However, even Bones Where bones are used as the starting material, the
these common steps can differ in duration and pH condition mode of manufacture will be the second parameter that wiIl
which can resuIt in significant differences in their inactivation ensure the safety of gelatin.
capacity. Manufacturers should at least conduct a process - Gelatin can be manufactured from bones from countries
evaluation based on the similarities of the collagen processing with a negligible, a controIled or an undetermined BSE
steps, as compared to known inactivation steps in the risk (Categories A, B or C) sourced in accordance with
manufacture of geIatin, in order to support the safety of the the conditions described in section 6-2 under The source
producto In addition to processing, differences also exist in material used, using the acid, alkaline or heatlpressure
the final use of the material and, consequendy, in their risk manufacturing process.
assessment, while gelatin is widely used for oral
administration, many collagen applications are in the form of - The manufacturing process shall be taken into
surgical implants. This aspect should also be considered in consideration when performing the risk assessment as
the final risk assessment. described in Section 4 of this chapter. Both the acid and
the alkaline manufacturing methods have shown similar
6-2 Gelatin overall inactivation/removal of TSE infectivity in the
Gelatin is a natural, soluble protein, gelling or non-gelling, gelatin validation experirnents. Studies have shown that an
obtained by the partial hydrolysis of collagen produced from additional alkaline treatment (pH 13, 2 h) of the
bones, hides and skins of animals. bones/ossein further increases the TSE
For gelatin, documentation to demonstrate compliance with inactivation/removal capacity of the manufacturing
this chapter needs to be provided taking into account the process. Other processing steps such as filtration, ion
provisions listed in sections 3 to 5. In addition, consideration exchange chromatography and UHT sterilisation also
should be given to the following 25 . contributes to the safety of gelatin .
THE SO URCE MATERIAL USED - For a typical alkaline manufacturing process, bones are
Gelatin used in medicinal products can be manufactured finely crushed, degreased with hot water and
from bones or hides. demineralised with dilute hydrochloric acid (at a
minimum of 4 per cent and pH < 1.5) over a period of at
Hides as the starting material On the basis of current
least 2 days to produce the ossein. This is followed by an
knowledge, hides used for gel atine production represent a
alkaline treatment with saturated lime solution (pH at
safer source material as compared to bones. However, it is
least 12.5) for a period of at least 20 days.
highly recommended that measures should be put in place to
avoid cross-contamination with potentially infected materials - Bovine bones may also be treated by an acid process.
during procurement. The lirning step is then replaced by an acid pre-treatment
where the ossein is treated at pH < 3.5 for a minimum of
Bones as the starting material Where bones are used to
10 hours.
manufacture gelatin, the quality of the starting materials
needs to be controlled as an additional parameter to ensure - A "flash" heat treatment (sterilisation) step at 138 oC
the safety of the final product. Therefore, the following minimum for 4 s at least is applied to both acid and
should be applied. alkaline manufacturing process.
l. SkuIls and spinal cord shaIl be removed from the - In the heatlpressure process, the dried degreased crushed
colIected bones (raw/starting material) independent of bones are autoelaved with saturated steam at a pressure
the age or the country of origin of the catde. greater than 3 bar and a minimum temperature of
133 oC, for at least 20 min, followed by extraction of the
2. Vertebrae shalI be removed from the raw/starting
protein with hot water.
materials from catde over 30 months from countries
with a controIled or an undetermined BSE risk - The finishing steps are similar for the alkaline, acid and
(Categories B or C) . heatlpressure process and inelude extraction of the
gelatine, washing, filtration and concentration.
3. Gelatin for parenteral use should only be manufactured
from bones coming from countries with a negligible or a 6-3. BOVINE BLOOD AND BLOOD DERIVATIVES
controlled BSE risk (Category A and B, respectively) . Foetal bovine serum is commonly used in cell cultures.
'Gelatin for oral use can be manufactured from bones Foetal bovine serum should be obtained from foetuses
from countries with a negligible, a controIled or an harvested in abanoirs from healthy dams fit for human
undetermined BSE risk (Category A, B and C, consumption and the womb should be completeIy removed
respectively) . and the foetal blood harvested in dedicated space or area by
4. Gelatin shaIl be manufactured using one of the cardiac puncture into a elosed colIection system using aseptic
manufacturing methods described below. technique.
MANUFACTURING METHODS Newbom calf serum is obtained from calves under 20 days
Hides No specific measures with regard to the processing old and calf serum from animals under the age of 12 months.
conditions are required for gelatin produced from hides In the case of donor bovine serum, given that it may be
derived from animals less than 36 months old, the TSE
25 Based on the Opinion of the ScienLific Panel on Biological Hazards of the negative status of the donor herd shaIl be welI defined and
European Food Safety Authority on the 'Quantitative assessment of ¡he documented. In alI cases, serum shall be collected according
human BSE risk posed by gelaLine with respeet 10 residual BSE risk '. to specified protocols by personnel trained in these
The EFSA Journal, 312, (1-28) . hup: //www.efsa.europa.eu/EFSA / procedures to avoid cross-contamination with higher risk
efsa_locale-1178620753812_1178620776107.htm The requirements for tissues.
source material selection and manufacture are appropriate for oral or
parenteral gelatin for use in human and veterinary medicinal produets.
V -A600 Appendix XXII B 2014
For bovine blood and blood derivatives, documentation to is comparable, it is recommended that manufacturers
demonstrate compliance with this chapter needs to be undertake product-specific investigational studies.
provided taking into account the provisions listed in sections Investigations using biochemical assay may be sufficient if
3 to 5. In addition, consideration should be given to the there is scientific evidence that this assay correlates with
following. infectivity data. General guidance for investigational studies
TRACEABILITY on reduction of TSE agents has been outlined29 •
Traceability to the slaughterhouse must be assured for each Brain-derived spike preparations are appropriate for studies
batch of serurn or plasma. Slaughterhouses must have investigating the risk from brain-contaminated blood.
available lists of farros from which the animals are originated. 6-4 Tallow derivatives
If serurn is produced from living animals, records must be Tallow is fat obtained from tissues including subcutaneous,
available for each serum batch which assures the traceability abdominal and inter-muscular areas and bones. Tallow used
to the farros. as the starting material for the manufacture of tallow
GEOGRAPHICAL ORIGIN derivatives shall be 'Category 3 material or equivalent', as
Whilst tissue infectivity of BSE in cattle is more restricted definedjn Regulation (EC) No 1774/2002 ofthe European
than scrapie, as a precautionary measure bovine blood should Parliament and of the Council of 3 October 2002 laying
be sourced from Category A countries . Bovine blood from down health rules conceming animal by-products not
Category B countries is also acceptable provided that there is intended for human consumption.
no risk for cross-contamination of blood with brain material Tallow derivatives, such as glycerol and fatty acids,
from the slaughter of animals over 21 months 26 of age. manufactured from tallow by rigorous processes are thought
STUNNING METHODS unlikely to be infectious and they have been the subject of
If it is sampled from slaughtered animals, the method of specific consideration by CHMP and CVMP.For this
slaughter is of importance to assure the safety of the material. reason, such materials manufactured under the conditions at
It has been demonstrated that stunning by captive bolt least as rigorous as those given below shall be considered in
stunner with or without pithing as well as by pneumatic compliance for this chapter, irrespective of the geographical
stunner, especially if it injects air, can destroy the brain and originand the nature of the tissues from which tallow
disseminate brain material into the blood stream. Non- derivatives are derived. Examples of rigorous processes are:
penetrative stunning is no more considered as an altemative ---'- trans-esterification or hydrolysis at not less than 200 oC
to penetrative stunning because contamination of blood with for not les s than 20 min under pressure {glycerol, fatty
brain material has been demonstrated 27 . Negligible risk can acids and fatty acid esters production),
be expected from electro-narcosis28 , but this even does not - saponification with 12 M NaOH (glycerol and soap
provide strict safety beca use, when unsuccessful, animals may production) :
have to be additionally stunned. The stunning methods must - batch process: at not les s than 95 oC for not less than 3 h,
therefore be described for the bovine blood collection
- continuous process : at not less than 140 oC, under
process .
pressure for not less than 8 min, or equivalent,
Whenever a risk of cross-contamination of blood with brain
- distillation at 200 oC.
cannot be avoided at routine slaughtering in countries with a
controlled BSE risk (Category B), safety measures·such as Tallow derivatives manufactured according to these
restriction of the age of the cattle andlor reduction of conditions are unlikely to present any TSE risk and shall
infectious agents during manufacture have to be applied. therefore be considered compliant with this chapter.
AGE Tallow derivatives produced using other conditions must
demonstrate compliance with this chapter.
For countries with a controlled BSE risk (Category B), a
precautionary age limit of 21 months shall apply for bovine 6-5 Animal charcoal
blood or blood derivatives where no significant reduction of Animal charco al is prepared by carbonisation of animal
TSE agents can be assumed from manufacture. Anage limit tissues, such as bones, using temperatures higher than
of 30 months is considered .gufficient for blood derivatives 800 oc. Unless otherwise justified, the starting material for
where significant reduction of TSE agents can be the manufacture of animal charcoal shall be Category 3
demonstrated as described below. material or equivalent, as defined in Regulation (EC) No
REDUCTION OF TSE AGENTS DURING MANUFACTURE 177 4/2002 of the European Parliament and of the Council of
For blood' derivadves, the capacity of the manufacturing 3 October 2002 laying down health rules concerning animal
process to reduce/eliminate TSE agents should be estimated by-products not intended for human consumption.
from investigational studies. The estimation may be based on Irrespective of the geographical origin and the nature of the
published data or in house data whenever it can be shown tissue, for the purpose of regulatory compliance, animal
that such data is relevant to the specific manufacturing charcoal shall be considered in compliance with this chapter.
process. If it cannot be concluded that the reduction capacity Charco al manufactured according to these conditions is
unlikely to present any TSE risk and shall therefore be
26 Opinion of che Scien!ific Panel on Biological Hazards pn the assessmen! of considered compliant with this chapter. Charcoal produced
che age limit in catTle for che removal of cenain Specified Risk Macerials using other conditions must demonstrate compliance with
(SRM). Question No EPSA-Q-2004-146, adopced on 28 April2005 this chapter.
27 The cissue classification cables are based upon che mosc recen! W'HO
Guidelines on Tissue Infectivity Distribution in Transmissible Spongiform
Encephalopathies (2010)
hup: //www. who.int/bloodproducts /tablestissueinfectivity.pdj
28 Repon of the EFSA Working Group on BSE risk from dissemination of
brain particles in blood and carcass. Question No EPSA-Q-2003-112,
adopted on 21 October 2004, hup://www.efsa.europa.eu/en/sciencebiohaz/ 29 Guideline on che investigacion of manufacturing process for plasma-derived
biohaz_opinions/opinion_annexes/733.html medicinal products with regard Lo vCJD risk CPMP/BWP/5 136/03.
2014 Appendix XXII B V -A60 1
Table 5.2.8.-1. - Concept for acceptance of bovine blood/sera and derivatives

Product Poetal Donor calf Adult Calfserum Adult Adult Adult Adult
bovine serum bovine bovine bovine bovine bovine
serum donor serum / serum / serum serum
serum plasma plasma derivative derivative
/ serum
derivative
Geographical Cat. A Cat. A Cat. A Cat. A Cat. A Cat. B Cat. A Cat. B
origin of cattle and B and B and B1 and B
Age of cattle unborn < 1 year < 36 < 1 year No limit < 21 No limit < 30
months months 2 months
Slaughter- No risk of cross-contamination Risk of cross-contamination
ing/ cross-con-
tamination of
blood with CNS
material
Demonstration No No Yes 3
of Prion
reduction during
manufacture
1. When sourced in Category B countries. cattle should be from well·defined and documented herds.
2. A higher age may be allowed if cross·contamination of blood with CNS material can be clearly ruled out (e.g. halal slaughter).
3. Demonstration of prion reduction may not be required if cross·contamination of blood with CNS material can be c1early ruled out
(e.g. hala! s!aughter).
6-6 Milk and milk derivatives risk and shall therefore be considered compliant with this
In the light of the current scientific knowledge and chapter.
irrespective of the geographical origin, bovine milk is unlikely ~ The milk is sourced from healthy animals in the same
to present any risk of TSE contamination30 . conditions as milk collected for human consumption, and
Certain materials, including lacto se, are extracted from whey, - no other ruminant materials, with the exception of calf
the spent liquid from cheese production following rennet, are used in the preparation of such derivativ:es
coagulation. Coagulation can involve the use of calf rennet, (e.g. pancreatic enzyme digests of casein).
an extract from abomasum, or re=et derived from other Milk derivatives produced using other processes or rennet
ruminants. The CHMP/CVMP have performed a risk derived from other ruminant species must demonstrate
assessment for lactase and other whey derivatives produced compliance with this chapter.
using calf rennet and concluded that the TSE risk is
negligible if the calf rennet is produced in accordance with 6-7 Wool derivatives
the process described in the risk assessment report3 !. Derivatives of wool and hair of ruminants, such as lanolin
The conclusion was endorsed by the SSC 32 which has also and wool aleohols derived from hair shall be considered in
performed an assessment of the TSE risk of rennet in compliance with this chapter, provided the wool and hair are
general 33 . sourced from live animals.
Bovine milk derivatives manufactured according ta the Wool derivatives produced from wool which is sourced from
conditions described below are unlikely to present any TSE slaughtered animals declared "fit for human consumption"
and the manufacturing process in reIation to pH,
temperature and duration of treatrnent meets at least one of
30 For mi/k and milk derivatives ¡rom small ruminanlS, please see EFSA the stipulated processing conditions listed below are unlikely
opinion on Question No EFSA-Q-2008-310, adopted on 22 October 2008, to present any TSE risk and shall therefore be considered
hup: //www.efsa.eurapa.eulenlscdocslscdocl849.htm
compliant wirh this chaprer.
31 Committee for Medicinal Products for Human Use and ilS Biologics
Working Parry conducted a risk and regulatory assessnzent of lactase prepared ~ Treatment at pH2': 13 (initial ; corresponding to a NaOH
using calf rennet. The risk assessment inc/uded the source of ¡he animals, the concentrarion of ar leasr 0.1 M NaOH) at 60 cC for ar
excision of ¡he abomasums and ¡he availability of well-defined qualiry leasr 1 h. This occurs normally during the reflux stage of
assurance procedures. The qualiry of any nn/k replacers used as feed for ¡he the organic-alkaline treatrnent.
animals fronz which abomasums are obtained is particularly important.
- Molecular distilIarion at 2': 220 oC under reduced
The report can be found on
pressure. Wool derivarives produced using other
hup: Ilwww.enza.europa. eulpdfslhwnanlpresslpusI057102.pdf
32 Provisional statement on the safety of calf-derived rennet for the
conditions musr demonstrare compliance wirh this
manufacture of lactose, adopted by the SSC al its meeting of 4-5 April 2002 chapter.
(hIlP: Ilec. europa. eulfood!jslsclssclout255_en. pdj)
33 The SSC issued an opinion on ¡he safety of animal rennet in regard 10
¡'Ísks fronz animal TSE and BSE in particular, adopted at its meeting of
16 May 2002 (hIlP: llec. europa.eulfoodlfslsclssclout265jn.pdj)
Category lA: High-infectivity tissues
Tissue Cattle Sheep and goats Elk and deer

BSE Sera pie CWD
Infeetivityl PrPTSE Infectivityl PrpTSE Infeetivityl PrpTSE
Brain + + + + + +
Spinal cord + + + + NT +
Retina + NT NT + NT +
Optic nerve 2 + NT NT + NT +
Spinal ganglia + + + + NT +
Trigeminal ganglia + + NT + NT
3 +
Pituitary gland NT + NT +
3
Dura mater NT NT NT NT NT NT
6-8 Amino acids Annex : Major Categories of Infectivity

Amino acids can be obtained by hydrolysis of materials from The tables below are taken from the WHO Guidelines on
various sources. Tissue Infectivity Distribution in Transmissible Spongiform
Unless otherwise justified, the starting material for the Encephalopathies (20 10) .
manufacture of amino acids shall be 'Category 3 material or Data entries are shown as follows:
equivalent', as defined in Regulation (EC) No 1774/2002 of + Presence of infectivity or PrPTSE
the European Parliament and of the Council of 3 October Absence of detectable infectivity or PrPTSE
2002 laying down health rules conceming animal by-products NT Not tested
not intended for human consumption. NA Not applicable
Amino acids prepared using the following processing Uncertain interpretation
conditions are unlikely to present any TSE risk and shall be () Limited or preliminary data
considered compliant with this chapter : [] Infectivity or PrPTSE data based exc1usively on
- amino acids produced from hides and skins by a process bioassays in transgenic
which involves exposure of the material ro a pH of 1 to 2,
followed by a pH of > 11, followed by heat trearment at
140 oC for 30 min at 3 bar,
- the resulting amino acids or peptides must be filtered after
production, and
- analysis is performed using a validated and sensitive
method ro control any residual intact macromolecules,
with an appropriate limit seto
Amino acids prepared using other conditions must
demonstrate compliance with this chapter.
6-9 PEPTONES
Peprones are partial hydrolysates of protein, achieved by
enzymic or acid digestion. They are used in microbiological
culture media ro support the nutritional requirements of
micro-organisms, which might be used as seed stocks or in
industrial scale fermentations for the production of human
and veterinary medicinal products, inc1uding vaccines. There
is considerable interest in the use of vegetable protein as an
altemative ro animal sourced protein. However :
- where gelatin is used as the protein source material,
reference is made to section 6-2 Gelatin, of this chapter,
-- where casein is used as the protein source material,
reference is made to section 6-6 Milk and mil k derivatives,
of this chapter,
- where tissue of TSE-relevant animal species is the protein
source material, the tissue must be sourced from animal s
fit for consumption (see section 3-2 Source animal s, of
this chapter) with a maximum age of 30 months old for
cattle from countries with a controlled BSE risk (Category
B). The age of animals is of minimal concem for animal s
from countries with a negligible BSE risk (Category A) .
Category lB: Lower-infedivity tissues

BSE Scra~ie CWD
Infectivityl PrP TSE
Infectivityl PrpTSE Infectivityl PrpTSE
Peripheral nervous system
Peripheral nerves [+] + + + NT +

4
Autonomic ganglia NT + NT + NT +
Lymphoreticular tissues
Spleen + + NT +
Lymph nodes + + NT +
Tonsil + + + NT +
Nictitating + [+] + NT +
membrane
Thymus NT + + NT
Alimentary tract5
Oesophagus NT [+] + NT +
6
Fore-stomach NT [+] + NT +
(ruminants only)
Stomach/ abomasum NT [+] + NT +
Duodenum [+] + NT +
7
Jejunum + [+] + NT NT
Ileum 7 + + + + NT +
Appendix NA NA NA NA NA NA
Colon/ caecum 7 + + NT +
Rectum NT NT NT + NT +
Reproductive tissues
PlacentaS NT + + NT
Ovary 3 NT NT
Uterus 3 NT NT
Other tissues
Mammary NT + NT NT
gland/ udder9
Skin:1, 1O NT + [+] [+]
Adipose tissue NT NT NT [+] NT
Heartjpericardium NT NT NT +
Lung NT NT +
Liver3 NT + NT
Kidnel,11 [+] + NT +
Adrenal [+] + + NT +
3 +
Pancreas NT + NT NT
Bone marrow 12 [+] NT + NT NT
13 +
Skeletal muscle [+] NT [+] [+]
14
Tongue NT [+] + NT
Blood vessels NT NT + NT
Nasal mucosa 15 NT + + NT +
Salivary gland NT + NT
Cornea 16 NT NT NT NT NT NT
Body fluids, secretion and excretions
CSF NT + NT NT
Blood 17 + +
Saliva NT NT NT + [-]
Milk 18
+ [+] NT NT
Urine 19 NT -[+] [+]
19 - +1
Category IC: Tissues with no detectable infectivity

BSE Scral!ie CWD
Infectivity! PrpTSE Infectivity! PrpTSE Infectivityl PrpTSE
Reproductive tissues
Testis NT NT
Prostate/ Epididy- NT NT
mis/Seminal vesicle
Semen NT NT NT
Placenta fluids NT NT NT NT NT
Foetus 2O
NT NT (-)
Embryos2O NT NT NT NT
Musculo-skeletal tissues
Bone NT NT NT NT NT
Tendon NT NT NT NT NT
Other tissues
Gingival tissues NT NT NT NT NT NT
Dental pulp NT NT NT NT NT NT
Trachea NT NT NT NT
Thyroid gland NT NT NT NT
Body fluids, secretions and excretions
Colostrum 2! (-) (?) NT NT NT
2
Cord blood ! NT NT NT NT NT
Sweat NT NT NT NT NT NT
Tears NT NT NT NT NT NT
Nasal mucus NT NT NT NT NT NT
Bile NT NT NT NT NT NT
1. Infectivity bioassays of human tissues have been conducted in either primates or mice (or both), bioassays of cattle tissues have
been conducted in either cattle or roice (or both), and most bioassays of sheep and/or goat tissues have be en conducted only in miee.
In regard to sheep and goats not all results are consistent for both speeies, for example, two goats (but no sheep) have eontracted BSE
naturally [Eurosurveillance, 2005, Jeffrey et al., 2006]. Similarly, most of the results described for CWO were derived from studies in
deer, and findings may not be identical in elk or other cervids.
2. In experimental models of TSE, the optic nerve has been shown to be a route of neuroinvasion, and contains high titres of infectivity.
3. No experimental data about infectivity in pituitary gland or dura mater in humans with all forms of human TSE have been reported,
but eadaveric dura mater patches, and growth hormone derived from cadaveric pituitaries have transmitted disease to hundreds of
people and therefore must be included in the category of high-risk tissues. PrPTSE was detected by immunoblot in the dura mater of a
vCJO patient who died in the US after an unusually long incubation period (see also Table lB for other positive tissues: skin, kidney,
liver, pancreas, ovary and uterus) [Notari et al., 2010]. It must be mentioned that earlier studies of numerous cases examined in the
UK reported all of these tissues to be negative [Ironside et al., 2002, Head et al., 2004].
4. In cattle, PrPTSE is reported to be inconsistently present in the enteric plexus in the distal ileum, but immunohistochemical
examination of tissues from a single 'fallen stock' case of BSE in Japan suggested (albeit equivocally) involvement of myenteric
plexuses throughout the small and large intestine [Kimura and Haritani, 2008J.
5. In vCJO, PrPTSE is limited to gut-associated lymphoid and nervous tissue (mucosa, rouscle, and serosa are negative).
6. Ruminant fore stomachs (reticulum, rumen, and omasum) are widely consumed, as is the true stomach (abomasum). The abomasum
of cattle (and sometimes sheep) is also a source of rennet.
7. When a large BSE oral dose was used to infect cattle experimentally, infectivity was detected in the jejunum and the il eo-caecuro
junction in Tg roice overexpressing PrP [courtesy of Or M GroschupJ. PrPTSE was detected at low incidence in lymphoid tissue of
ileum [Terry et al., 2003] and has been detected at an even lower frequency in jejunal lymphoid tissue of cattle similarly infected by
the oral route [EFSA, 2009J.
8. A single report of transmission of sporadic CJO infectivity from hum an placenta has never been confirmed and is considered
improbable.
9. PrPTSE has been detected in scrapie-infected sheep with chronic mastitis, but not from infected sheep without mastitis [Ligios
et al., 2005J.
10. Studies in hamsters orally infected with scrapie revealed that PrpTSE deposition in ski n was primarily located within small nerve
fibres. AIso, apical skin 'velvet' from the antlers of CWD-infected deer is reported to contain PrPTSE and infectivity [Angers et al., 2009J.
11. PrPTSE detected by immunocytochemistry in the renal pelvis of scrapie-infected sheep [Siso et al., 2006], and in lymphoid follicles
within connective tissue adjacent to the renal pelvis in CWD-infected mule deer [Fox et al., 2006J.
12. A single positive marrow in multiple transmission attempts from cattle orally dosed with BSE-infected brain [Wells et al., 1999,
Wells et al., 2005, Sohn et al., 2009J.
13. Muscle homogenates have not transmitted disease to primates from humans with sporadic CJO, or to cattle from cattle with BSE.
However, intra-cerebral inoculation of a semitendinosus muscle homogenate (including nervous and lymphatic elements) from a single
cow with clinical BSE has transmitted disease to transgenic mice that overexpress PrP at arate indicative of trace levels of infectivity
[Buschmann and Groschup, 2005]. AIso, recent published and unpublished studies have reported the presence of PrPTS E in skeleta l
muscle in experimental rodent models of scrapie and vCJO [Beekes et al., 2005], in experimental and natural scrapie infections of
sheep and goats [Andreoletti et al., 2004J, in sheep orally dosed with BSE [Andreoletti, unpublished data] , and in humans with
sporadic, iatrogenic, and variant forms of CJO [Glatzel et al., 2003, Kovacs et al., 2004, Peden et al., 2006J. Bioassays of muscle in
transgen ic mice expressing cervid PrP have documented infectivity in CWD-infected mule deer [Angers et al., 2006J, and experiments
are underway to determine whether detectable PrP TSE in other forms ofTSE is also associated with infectivity.
14. In cattle, bioassay of infectivity in the tongue was negative, but the presence of infectivity in palatine tonsil has raised concern
about possible infectivity in lingual tonsillar tissue at the base of the tongue that may not be removed at slaughter [Wells et al.,
2005, EFSA, 2008]. In sheep naturally infected with scrapie, 7 of 10 animals had detectable PrPTSE in the tongue [Casalone et al.,
2005, Corona et al., 2006].
15. Limited chiefly to regions involved in olfactory sensory reception.
16. Because only one case of iatrogenic CJO has been certainly attributed to a corneal transplant among hundreds of thousands of
recipients (one additional case is considered probable, and another case only possible), cornea has been categorized as a lower-risk
tissue, other anterior chamber tissues (len s, aqueous humour, iris, conjunctiva) have been tested with a negative result both in vCJO
and other human TSEs, and there is no epidemiological evidence that they have been associated with iatrogenic disease transmission.
17. A wealth of data from studies of blood infectivity in experimental rodent models of TSE have been extended by recent studies
documenting infectivity in the blood of sheep with naturally occurring scrapie and in sheep transfused with blood from BSE-infected
cattle [Houston et al., 2008], of deer with naturally occurring CWD [Mathiason et al., 2006], and (from epidemiological observations)
in the red cell fraction (which includes significant amounts of both plasma and leukocytes) of four blood donors in the pre-clinical
phase of vCJO infections [reviewed in Brown, 2006, Hewitt et al., 2006J. Plasma Factor VIII administration has also be en potentially
implicated in a subclinical case of vCJO in a haemophilia patient [Peden et al., 2010]. Blood has not been shown to transmit disease
from humans with any form of 'classical' TSE [Dorsey et al., 2009], or from cattle with BSE (including fetal calf blood). A number
of laboratories using new, highly sensitive methods to detect PrP TSE are reporting success ina variety of animal and human TSEs.
However, several have experienced difficulty obtaining reproducible results in plasma, and it is not yet clear that positive results
implya potential for disease transmissibility, either because of false positives, or of 'true' positives that are due to sub-transmissible
concentrations of PrpTSE. Because of these considerations (and the fact that no data are yet available on blinded testing of specimens
from naturally infected humans or animals) the expert group felt that it was still too early to evaluate the validity of th ese tests with
sufficient confidence to permit either a negative or positive conclusion.
18. Evidence that infectivity is not present in milk from BSE-infected bovines includes temporo-spatial epidemiologic observations
failing to detect maternal transmission to calves suckled for long periods, clinical observations of over a hundred calves suckled by
infected cows that have not developed BSE, and experimental observations that milk from infected cows reared to an age exceeding
the minimum incubation period has not transmitted disease when administered intra-cerebrally or orally to mice [Middleton and
Barlow, 1993, Taylor et al., 1995]. AIso, PrPTSE has not been detected in milk from cattle incubating BSE following experimental oral
challenge [SEAC, 2005J. However, low levels (lJg to ng/ L) of normal PrP have been detected in milk from both animals and humans
[Franscini et al., 2006J. Prp TSE has been detected in the mammary glands of scrapie-infected sheep with chronic mastitis [Ligios et al.,
2005J, and very recently it has been reported that milk (which in sorne cases also contained colostrum) from scrapie-infected sheep
transmitted disease to healthy animals [Konold et al., 2008, Lacroux et al., 2008].
19. A mixed inoculum of urine and faeces from naturally infected CWD deer did not transmit disease during an 18-month observation
period after inoculation of healthy deer with a heterozygous (96 G/ S) PRNP genotype [Mathiason et al., 2006]. However, recent
bioassays in Tg mi ce have transmitted disease from both urine [HaJey et al. , 2009J and faeces [Tamgüney et al., 2009J. In addition, mice
with lymphocytic nephritis that were experimentally infected with scrapie shed both PrPTSE and infectivity in urine, when bioassayed in
Tg mice [Seegeret al., 2005J. Very low levels of infectivity have also been detected in the urine (and histologically normal kidneys) of
hamsters experimentally infected with scrapie [Gregori and Rohwer, 2007, Gonzalez-Romero et al., 2008J. Finally, in an experimental
scrapie-hamster model, oral dosing resulted in infectious faeces when bioassayed in Tg mice over-expressing PrP [Safar et al., 2008J.
20. Embryos from BSE-affected cattle have not transmitted disease to mice, but no infectivity measurements have been made on fetal
calf tissues other than blood (negative mouse bioassay) [Fraser and Foster, 1994J. Calves born of dams that received embryos from
BSE- affected cattle have survived for observations periods of up to seven years, and examination of the brains of both the unaffected
dams and their offspring revealed no spongiform encephalopathy or PrPTSE [Wrathall et al., 2002].
21. Early reports of transmission of sporadic CJD infectivity from human cord blood and colostrum have never been confirmed and
are considered improbable. A bioassay from a cow with BSE in transgenic mice over-expressing bovine PrP gave a negative result
[Buschmann and Groschup, 2005J, and PrPTSE has not been detected in colostrum from cattle incubating BSE following experimental
oral challenge [SEAC, 2005J.
2014 Appendix XXIII B V -A607
Appendix XXIII
Weights and Measures
B. Conversion Tables for Commonly Used Units
The folIowing tables are included for the convenience of users. Table 23-5 gives the conversions from parts per million,
Table 23-6 gives commonly used concentrations and their conversions .
Table 23-5 Parts Per Million (ppm) Conversions
Parts per billion Parts per núllion ILg/g Fraction Decimal Percentage w/w
(ppb) (ppm)
0.001 0.001 1/1 000 000 000 0.000000001 0.0000001 %

10 0.01 0.01 1/100 000 000 0.00000001 0.000001 %
100 0.1 0.1 1/10000000 0.0000001 0.00001 %
1000 1 1/1 000000 0.000001 0.0001%
5000 5 5 5/1 000000 0.000005 0.0005%
10000 10 10 1/100000 0.00001 0.001 %
20 20 2/100 000 0.00002 0.002%
50 50 5/100 000 0.00005 0.005%
100 100 1/10 000 0.0001 0.01%
200 200 2/10000 0.0002 0.02%
1000 1000 1/1000 0.001 0.1%
5000 5000 5/1000 0.005 0.5%
Table 23-6 Concentration Conversions
ILg/rnl mg/mI g/mI g/lOO mI

(% w/v)
1 0.001 0.000001 0.0001

10 0.01 0.00001 0.001
20 0.02 0.00002 0.002
50 0.05 0.00005 0.005
100 0.1 0.0001 0.01
200 0.2 0.0002 0.02
500 0.5 0.0005 0.05
1000 0.001 0.1
10000 10 0.01
V -A608 Appendix XXIV 2014
Appendix XXIV
Abbreviations
ATCC* American Type Culture CoIlection.
BPCRS British Pharmacopoeia Chemical Reference Substance (see Appendix 1 E).
BRP Biological reference preparation (see Appendix 1 E).
BS British Standard.
CCID so CeIl culture infective dose (the dose of the micro-organism that infects 50% of the ceIl cultures inoculated).
CIP* CoIlection de Bactéries de l'Institut Pasteur.
CRS Chemical reference substance (se e Appendix 1 E).
DNA Deoxyribonucleic acid.
EIDso Egg embryos infective dose (the dose of the micro-organism that infects 50% of the embryonated eggs inoculated).
EPBRP European Pharmacopoeia Biological Reference Preparation (see Appendix 1 E).
EPCRS European Pharmacopoeia Chemical Reference Substance (se e Appendix 1 E).
FIP Intemational Pharmaceutical Federation.
g Acceleration due to gravity.
HN Human irnmunodeficiency virus
IDso Infective dos e 50 (the dose of the micro-organism that infects 50% of the animals inoculated).
IMI* Commonwealth M ycological Institute.
IP* CoIlection Nationale de Cultures de Microorganismes (CNCM) .
ISO Intemational Organization for Standardization.
IU Intemational Unit
IUPAC Intemational Union of Pure and Applied Chemistry.
LDso Lethal dose 50 (the dose of the preparation or organism that kiIls 50% of the animals inoculated).
MID Minimum infective dose.
MLD Minimum lethal dose.
MRI Magnetic resonance imaging.
NCIMB* National CoIlection of Industrial and Marine Bacteria.
NCPF* National CoIlection of Pathogenic Fungi.
NCTC* National Collection of Type Cultures.
NCYC* N ational Collection of Yeast Cultures.
PD so Protective dose 50 (the dose of the preparation that protects 50% of the animals inoculated) .
ppm Parts per million by weight.
SI Intemational System of Units.
SNso Serum neutralising dose 50 (the amount of serum that will protect 50% of the cultures against the specified amount
ofvirus).
SSI Statens Serum Institut (Copenhagen) .
THM Traditional Herbal Medicine.
THMP Traditional Herbal M edicinal Product.
VS Volumetric solution (se e Appendix 1 B) .
~kat .Microkatal: the enzyme activity that, under defined conditions, produces 1 micromole of the reaction product per
second or consumes one micromole of the reaction substrate per second .
• S¡rains of che micro-organisms referred ro in the Pharmacopoeia may be obtained from:

ATCC American Type Culture Collection, 10801 University Boulevard, M anassas, Virginia 20110-2209, USA.
CIP Collection de Baaéries de l'Insútut Pas¡eur, BP 52, 25 Rue du Dr-Roux, F-75724, Paris Cedex 15, France.
IMI Imemational Mycological Ins¡üute, Bakeham Lane, Surrey TW2 0 91Y, England.
IP Service de la Collection Nationale de Culture de Microorganismes (CNCM), Institut PasteU/; 25 Rue de DI' Roux, F-75724, Pans Cedex 15 Frallce.
NCIMB National Collecúon of Industrial and Mmine Bacteria Ltd, 23 St Machar Drive, Aberdeen, AB24 3RY, Scocland.
NCPF Nacional Collection of Pachogenic Fungi, London School of Hygiene and Tropical Medicine, Keppel Sereet, London WCIE 7HT, England.
NCTC NaúOlzal Collection of Type Cultures, Central Public Heallh LaboratOlY, Colindale Avenue, London NW9 5HT, England.
NCYC National Col/ection of Yeas¡ Cultures, AFRC Food Research Imtitute, Colney Lane, Norwich NR4 7UA, England.
2014 Appendix XXV V-A609
Appendix XXV
Names, Symbols and Atomic Weights of Elements
The following atomic weights are those published in 2001 by the Intemational Union ofPure and Applied Chemistry (Pure
Appl. Chem. 2003, 75, 1107).
E lement Symbol Atomic Weight Element Symbol Atomic Weight
Actinium Ac [227] Molybdenum Mo 95.94

Aluminium Al 26.981538 Neodymium Nd 144 .24
Americium Am [243] Neon Ne 20.1797
Antimony Sb 121.760 Neptunium Np [237]
Argon Ar 39.948 Nickel Ni 58.6934
Arsenic As 74.92160 Niobium Nb 92.90638
Astatine At [210] Nitrogen N 14.0067
Barium Ba 137 .327 Nobelium No [259]
Berkelium Bk [247] Osmium Os 190.23
Beryllium Be 9.012182 Oxygen O 15.9994
Bismuth Bi 208 .98038 Palladium Pd 106.42
Bohrium Bh [264] Phosphorus P 30.973761
Boron B 10.811 Platinum Pt 195.078
Bromine Br 79.904 Plutonium Pu [244]
Cadmium Cd 112.411 Po1onium Po [209]
Caesium Cs 132.90545 Potassium K 39.0983
Calcium Ca 40.078 Praseodymium Pr 140.90765
Califomium Cf [251] Promethium Pm [145]
Carbon C 12.0107 Protactinium Pa 231.03588
Cerium Ce 140.116 Radium Ra [226]
Chlorine CI 35.453 Radon Rn [222]
Chromium, Cr 51.9961 Rhenium Re 186.207
Cobalt Co 58.933200 Rhodium Rh 102.90550
Copper Cu 63.546 Roentgenium Rg [272]
Curium Cm [247] Rubidium Rb 85.4678
Darmstadtium Ds [281] Ruthenium Ru 101.07
Dubnium Db [262] Rutherfordium Rf [261]
Dysprosium Dy 162.500 Samarium Sm 150.36
Einsteinium Es [252] Scandium Se 44.955910
Erbium Er 167.259 Seaborgium Sg [266]
Europium Eu 151.964 Selenium Se 78.96
Fermium Fm [257] Silicon Si 28.0855
Fluorine F 18.9984032 Silver Ag 107 .8682
Francium Fr [223] Sodium Na 22.989770
Gadolinium Gd 157.25 Strontium Sr 87.62
Gallium Ga 69 .723 Sulfur S 32.065
Germanium Ge 72.64 Tantalum Ta 180.9479
Gold Au 196.96655 Technetium Te [98]
Hafnium Hf 178.49 Tellurium Te 127.60
Hassium Hs [277] Terbium Tb 158.92534
Helium He 4.002602 Tha!lium TI 204.3833
Holmium Ho 164.93032 Thorium Th 232.0381
Hydrogen H 1.00794 Thulium Tm 168.93421
Indium In 114.818 Tin Sn ' 118 .71
Iodine I 126.90447 Titanium Ti 47.867
Iridium Ir 192.217 Tungsten W 183.84
Iron Fe 55.845 Ununbium Uub [285]
Krypton Kr 83.798 Ununhexium Uuh
Lanthanum La 138 .9055 Ununoctium Uuo
Lawrencium Lr [262] Ununquadium Uuq [289]
Lead Pb 207.2 Uranium U 238 .02891
Lithium Li [6.941] Vanadium V 50.94 15
Lutetium Lu 174.967 Xenon Xe 131.293
Magnesium Mg 24. 3050 Ytterbium Yb 173.04
Manganese Mn 54.938049 Yttrium Y 88.90585
M eitnerium Mt [268] Zinc Zn 65.409
Mendelevium Md [258] Zirconium Zr 91.224
Mercury Hg 200.59
Suppletnentary Chapters
Supplementary Chapters contain no standardsJ tests or assays nor any other mandatory
specifications with respect ro any Pharmacopoeial article. They comprise explanarory and
other ancillary texts and are provided for the assistance and information of usen of the
Pharmacopoeia.
2014 V-A613
Contents of the Supplementary Chapters

SUPPLEMENTARY CHAPTER 1 A615
Basis of Phannacopoeial Requirements A615
A. Control of Impurities A616
B. Polymorphism A619
C. Bacterial Endotoxin Testing A620
D. Excipients A624
E. Dissolution Testing of Solid Oral Dosage Fonns A624
F. Declaration of Content A630
G. Labelling A631
H. Biological Assays and Tests A63I
J. Efficacy of Antimicrobial Preservation A633
K. Stereochemistry A634
L. Microbiological Assay of Antibiotics A634
M. Microbial Contamination A636
N. Particulate Contamination A637
O. Inhaled Products A638
SUPPLEMENTARY CHAPTER II A639
Names of Medicinal Substances and Preparations A639
A. Changes in Monograph Titles A639
B. Monograph Titles for Formulated Preparations A639
C. Structures and Nomenclature of Substances of Natural or
Semi-synthetic Origin A64I
SUPPLEMENTARY CHAPTER III A647
Phannacopoeial Organisation A647
Al. Contact Points A647
A2. Expert Advisory Groups A647
B. Monograph Development: Mechanism A648
C. Monograph Development: Guidance to Manufacturers A649
D. Monograph Development: Methods of Analysis A651
E. British Phannacopoeia Chemical Reference Substances (BPCRS) A653
F. Validation of Analytical Procedures A653
SUPPLEMENTARY CHAPTER IV A656
European Phannacopoeia A656
A. Membership of the European Phannacopoeia Commission A656
B. Dates of Implementation A657
C. Certification Scheme A657
D. Residual Solvents A658
E. Alcoholimetric Tables A667
F. Phannacopoeial Harmonisation A674
G. Statistical Analysis of Results of Biological Assays and Tests A678
H. MateriaIs Used in Chromatographic Tests A712
1. Chromatograms for Infonnation (Transferred to BP website
www.phannacopoeia.com)
V-A614 2014
J. Control of Impurities in Substances for Pharmaceutical U se A713

K. Characters Section in Monographs A716
L. Altemative Methods for Control of Microbiological Quality A716
M. Reference Standards A728
N. Gene Transfer Medicinal Products for Human Use A732
O. Functionality-related Characteristics of Excipients A745
P. Guidelines for Using the Test for Sterility A746
Q. Metal Catalyst or Metal Reagent Residues A747
SUPPLEMENTARY CHAPTER V A752
Unlicensed Medicines A752
A. Monograph Selection: Unlicensed Medicines A754
B. Preservative-free Unlicensed Medicines A754
C. Bioequivalence of Oral Liquids A755
D. Storage and Stability of Unlicensed Medicines A755
E. Extemporaneous Preparations A756
SUPPLEMENTARY CHAPTER VI A757
Pharmacopoeial Quantitative Analysis A757
A. Pharmacopoeial Calculations A757
B. Titrimetric Analysis A759
C. Indicator Colour Changes A760
SUPPLEMENTARY CHAPTER VII A763
Traditional Herbal Medicines A763
SUPPLEMENTARY CHAPTER VIII A764
Materials for use in the Manufacture of Homoeopathic Preparations A764
SUPPLEMENTARY CHAPTER IX A765
Similar Biological Medicinal Products A765
2014 Supplementary Chapter 1 V -A615
manufacturer will need to apply more stringent test limits at

Supplementary the time of release of a batch of the product or material in
order to ensure compliance. As stated in the General
Chapter 1 Notices, a manufacturer may assure himself that the
requirements of the Pharrnacopoeia will be met by means
other than routinely perforrning all of the tests prescribed in
Basis of Pharmacopoeial Requirements the Pharrnacopoeia. It is emphasised that the circumstances
under which, and the frequency with which, tests of the
Introduction Pharrnacopoeia should be perforrned by a manufacturer as
This chapter provides explanatory text and guidance on the pan of his overall quality assurance are ultimately matters for
phannacopoeial approach to a range olsubjects. agreement berween the manufacturer and the competent
Separate, lettered sections of this chapter explain the current authority.
approach to a particular aspect of pharrnacopoeial control 4. The requirements induded in a monograph, other than
and, where appropriate, give an indication of future any instructions given under the side-heading Production, are
developments. The British Pharrnacopoeia Commission's designed to provide the means by which an independent
policies continue to evolve and these sections will be updated judgement can be made as to the overall quality of a
as and when necessary to reflect further developments. particular artide. A manufacturer in possession of detailed
While these texts outline general policies that are adopted in knowledge of the manufacturing process may have no need
the Pharrnacopoeia, each monograph is considered to carry out certain tests. The example of sorne impurity tests
individually. Departures from the general rule are accepted in monographs for formulated preparations is discussed in
where justified and are accommodated by appropriate more detail in section A of this chapter. The methods
statements in the individual monographs. described in the Pharrnacopoeia must be robust because they
are intended to be used by analysts in a wide range of
In providing these texts to users of the Pharrnacopoeia it is
laboratories, sometimes on an infrequent basis.
emphasised that the specifications of the Pharrnacopoeia are
Understandably, a manufacturer may wish to use other
one facet of the overall control of the quality of medicinal
methods that may be more suitable for frequent use or
products and their constituents. Those con cerned with the
automation and is entitled to do so. However in the event of
manufacture of medicinal substances and those responsible
any doubt or dispute as to whether or not a material is of
for their incorporation into pharrnaceutical dosage forms
pharrnacopoeial quality, as the General Notice on Assays and
must also pay due attention to the requirements and
Tests makes c1ear, the methods of the Pharrnacopoeia alone
recommendations of other competent authorities. Within the
are authoritative.
European Community the information required for
marketing authorization is laid down in the relevant 5. This view of pharrnacopoeial requirements is also
Directives, Notice to Applicants and associated Notes for significant when considering the size of sample to be taken
Guidance available from the Commission of the European for test. In an overall programme designed to give assurance
Communities as the series 'The Rules Governing Medicinal of quality of a manufactured product, the statistical validity
Products in the European Community' . of any sampling programme must be beyond doubt.
The standards of the Pharrnacopoeia, on the other hand, are
Dialogue with users is an essential element of
intended to apply to the sample available, perhaps the
pharrnacopoeial development and the British Pharrnacopoeia
container of dispensed tablets provided to a patient in
Cornmission hopes that this chapter will provide insight into
accordance with a prescription. The Pharrnacopoeia requires
certain features of pharrnacopoeial requirements.
that twenty of those ·tablets should meet the test for
The Commission places great value on the assistance it
Uniforrnity of weight. A manufacturer establishing a sampling
receives from manufacturers and others with the necessary
and testing protocol designed to ensure ultimate compliance
knowledge to assist it in its work. It welcomes suggestions for
with the pharrnacopoeial requirements will need to operate at
improvement of published texts and constructive comment
a level designed to show with an acceptable degree of
on any issues of interest and concern to users.
confidence that any twenty tablets, taken at random from a
given batch, will meet the requirements.
General considerations
6. Pharrnacopoeial methods and Iimits are set with the
1. A proper understanding of the basis on which the
intention that they should be used as compliance
requirements of the Pharrnacopoeia are established is
requirements and not as requirements to guarantee total .
essential to the correct interpretation of the requirements.
quality assurance. An artide is not of pharrnacopoetal qualIty
2. The Pharrnacopoeia contributes significantly to the overall if any sample of the size stipulated in the monograph taken at
control of the quality of medicinal products and provides a any time during storage, distribution and use within the
publidy available statement concerning the quality that a accepted shelf-life fails to meet all of the requirements.
product or a component of a product is expected to meet at
7. Arising from this it may be useful to underline that
any time during its period of use. Pharmacopoeial
compliance of a product with pharrnacopoeial requirements
specifications are used within Iicensing systems and by
demands that the product meets all mandatory aspects of the
manufacturers, suppliers, purchasers and those acting on
appropriate monograph and that those requirements shall be
behalf of consuní.ers of medicinal products.
interpreted in the Iight of any relevant General Nonces.
3. A manufacturer must recognise that a product or material In certain cases individual requirements of particular tests
may be challenged at any time during its daimed period of may seem to be incompatible with those of other tests; where
use by the methods of the Pharrnacopoeia and that it must this is apparently the case such requirements have been
then comply with the pharrnacopoeial requirements. These framed intentionally. For example, the requirement for the
requirements allow for acceptable levels of change that may overall content of active ingredient in a tablet preparation, as
occur during storage and distribution and reject artides deterrnined on a powdered sample of twenty tablets, might
showing unacceptable levels of change. Frequently a be 95 .0 to 105.0% of the stated amount. Thus an assay
V -A616 Supplementary Chapter 1 A 2014
result of 96.0% would indicate compliance. For the

Uniformity of content test a further ten tablets might be
A. Control of Impurities
individually examined, each tablet being required to contain This section provides a guide lO the pharnzacopoeial approach lO
between SS and 115% of the mean value, with the possibility the control of impurities in medicinal substances and formulated
of a single exception between 75 and 125%. Thus if nine out preparations. Ocher guidance relevanc lO manufacturers is provided
of ten tablets fall within the range (assuming the mean 10 be in, for example, /CH guidelines on impurities in new drug
96.0%) S1.6 and 110.4% and the tenth falls within the range substances (Q3A) and new drug preparations (Q3B) and V/CH
72 .0 10 120.0% then the tablets examined comply with that guidelines on impuricies in new veterinary drug subscances (GLJ O)
requirement. For the Dissolution test each tablet examined and new vecen'nary medicinal products (GUI).
might be required 10 yield at least 70% of the labelled claim l. This section relates primarily to totally synthetic organic
into solution within 45 minutes. It has been suggested that medicinal substances and those substances obtained by
since a single outlier tablet might contain as little as 72.0% of synthetic modification of a naturally-produced precursor. It is
the labelled claim and yet still fall within the acceptance not necessarily applicable to other organic substances (e.g.,
limits for content, the requirements for dissolution should be those of plant or animal origin), inorganic substances and
relaxed 10 take this into account. In framing requirements, excipients.
however, the view is taken that it is neither realistic nor Certain additional information of specific relevance to
profitable 10 attempt 10 compound the results of various tests impurity control in the formulated preparation monographs
in this way. Each test in a pharmacopoeial monograph and of the British Pharmacopoeia is also provided.
the acceptance limit is therefore framed as an individual 2. The control provided by chemical tests limiting the levels
entity with requirements based on values encountered in of particular impurities or classes of impurities is often
practice; compliance with the monograph requires augmented by physical tests such as absorbance, specific
compliance with each and every test. optical rotation, melting point and clarity and colour of
S. The philosophy outlined aboye has an important bearing solution and, for a liquid, refractive index, boiling point range
on the construction of a monograph for the Pharmacopoeia. and weight per mL.
To achieve maximum benefit from the examination of a 3. Tests such as sulfated ash, heavy metals and loss on
product the recommended approach is that, wherever drying are non-specific but they contribute to an assurance of
possible, a variety of different analytical techniques should be the general quality of the material, the use of good
employed. The monographs of the British Pharmacopoeia pharmaceutical manufacturing practice in its production, the
are, therefore, usually constructed to use fundamentally avoidance of contamination especially by inorganic
different procedures for assay and for the examination for substances and the removal of volatile solvents. Typical limits
impurities. For a medicinal substance the general approach are 0.1 % for sulfated ash, lOor 20 ppm for h-eavy metals and
has been to employ spectrophotometric or other appropriate 0.5 % for loss on drying.
techniques for identification, chromatographic techniques for 4. Tests for purity are intended to provide appropriate
the control of impurities and a precise, albeit non-specific limitation of known potential or actual impurities rather than
method for assay. It has been held that this approach confers 10 provide against all possible impurities . The tests are not
greater confidence in the verification of the identity and necessarily designed to detect any adventitious contaminants
quality of the substance and in the detection of unexpected or adulteration. Material found to contain an impurity not
impurities than would be the case in using, for example, a detectable by means of the prescribed tests is not of
single stability-indicating liquid chromatographic method for o pharmacopoeial quality if the nature or amount of the
all three purposes. The British Pharmacopoeia Commission impurity found is incompatible with good pharmaceutical
recognises, however, that as chromatographic methods practice.
become more precise it will become increasingly possible to
5. Sorne medicinal substances are mixtures of closely related
use them for assay purposes thereby combining precision
compounds. Where these components have similar activity
with specificity and economising upon analytical effort and
they are not usually regarded as impurities and may indeed
time. For dosage forms this concept has already been
contribute 10 the result obtained in the assay. Examples
adopted and more specific assay methods, such as those
include Erythromycin, Gentamicin Sulfate and Sodium
employing liquid chromatography, are being employed
Lauryl Sulfate. It may however be appropriate to control the
increasingly.
relative amounts of such components, e.g., gentamicins C¡,
9. A discussion of the basis of pharmacopoeial requirements C l ., C 2 , C 2a and C 2b in Gentamicin Sulfate in order 10
would be incomplete without reminding users that any article ensure batch to batch consistency for material from one
described by a name at the head of a monograph in the manufacturer and uniformity between supplies of the same
current edition of the Pharmacopoeia, whether or not it is substance from different manufacturers.
referred lO as 'BP', must comply with that monograph.
6. Many medicinal substances already on the market have
The name at the head of a monograph is 10 be interpreted in
been made available as racemic mixtures with little or
accordance with the General Notice on Titles. In particular,
nothing known about the biological activities of the separate
a formulated preparation that is labelled with a title that
isomers. This has been reflected in the monograph in the
includes the full nonproprietary name of the active
Pharmacopoeia and a test to show that the substance is the
ingredient, where this is not included in the title of the
racemic mixture has not usually been included unless it was
monograph, must also comply with the monograph. Thus,
known that at least one of the separate enantiomers was also
for example, a preparation labelled Labetalol Hydrochloride
available commercially. Nevertheless, with increasing concem
Tablets must comply with the monograph for Labetalol
by regula10ry authorities for substances to be made available
Tablets.
as single isomers, tests for enantiomeric composition will
become more common. When a medicinal substance is a
racemate, an indication is given by means of the graphic
formula [see also Supplementary Chapter 1 K;
Stereochemistry].
2014 Supplementary Chapter 1 A V-A617
Related Substances dibenzosuberone in Amitriptyline Tablets. Typical

7. It is usual to inc1ude a test for related substances in a wording is as follows:
monograph for a medicinal substance. These may be A ny SPO¡ corresponding 10 [x] in ¡he chromatogram obtained
manufacturing impurities (intermediates or by-products) or wüh solution (1) is not more intense ¡han ¡he pn'ncipal spot
degradation products or both. When preparation of a in the chromatogram obtained wi¡h solution (2). [Solution
monograph is initiated the manufacturer is asked to provide (1) contains ¡he substance being examined and solurion (2)
information concerning the nature of such impurities, the comains a named impurity [xj.]
reason for their presence, the amounts that may be lO. General tests for unnamed impurities It is unusual
encountered in material prepared under conditions of good for the Pharmacopoeia to require the absence of a visible
pharmaceutical manufacturing practice and the manner in spot in a thin-Iayer chromatogram or the absence of a peak
which proportions may vary on storage, together with an in a liquid chromatogram. Reasons for this inc1ude the
indication of the toxicity of any impurities in relation to that difficulty of interpreting and defining absence that is a
of the substance itself. Where there is only one manufacturer consequence of variations in the sensitivity of a method when
of a substance, pharmacopoeiallimits are set in the performed in different laboratories by different analysts . It is
knowledge that the level of impurities in production batches more usual to limit the levels of impurities. This may be
of the substance will have been accepted by the registration done in a simple test by comparison with a spot or peak
authority after a full consideration of the toxicity studies and obtained with a dilute solution of the substance being
c1inical trials carried out before the granting of a licence. examined. An example is Methyl Nicotinate:
Such studies and trials will have been carried out on material Any secondary spot in the chromatogram obtained wüh
with an impurity profile that is qualitatively and quantitatively solution (1) is nOl more intense ¡han the SpOl in ¡he
similar to that of subsequent production batches. chromatogram obtained with solution (2). [Solutions (1) and
Any subsequent changes to the manufacturing process by the (2) comain the substance being examined at high and low
original manufacturer or the introduction of material from concentrations respective1y.j
another manufacturer utilising a different route of synthesis
10.1 In the absence of evidence that the limit for a particular
will be subject to the need to demonstrate essential similarity
impurity needs to be set on the basis of its toxicity,
or to provide equivalent data to the relevant registration
control is often provided by a two-Ievel test requiring,
authority. In sorne cases a change in production or source
say, not more than one related substance at a nominal
may give rise to impurities that are not adequately controlled
concentration of up to 0.5% and any others at nominal
by the published pharmacopoeial monograph. Appropriate
concentrations of up to 0.1 %. The actual limits may be
revision of the monograph will be carried out provided that
chosen on the basis of batch data for material
the pharmacopoeial authority is notified of the need and that
manufactured in accordance with good pharmaceutical
it is supplied with the relevant information [se e paragraph 28
manufacturing practice and will take account of a
below] .
number of factors, inc1uding the dose regimen of the
8. Tests for related substances may be specific or general. substance and the number of impurities commonly
9. Specific tests for named impurities A specific test is presento An example is Oxetacaine:
inc1uded where a particular impurity arising from the Any secandary spot in ¡he chramatogram obtained with
manufacturing process or from degradation needs to be solutian (1) is not mOl"e intense ¡han the spot in the
limited on grounds of toxicity or for another special reason. chromatogram obtained with solution (2) (0.5%) and not
Where an impurity is known to be particularly toxic, this is more than one such spat is more intense than ¡he spot in the
taken into account in setting the limit; for example, a limit of chromatogram obtained with solution (3) (0.1 %) .
1 ppm is specified for hydrazine in the monograph for
Another example, with different limits, is Phenindione:
Povidone.
Any secandary spot in the chromatogram obtained with
9.1 Such specific tests usually employ a chromatographic or
solution (1) is not more imense than the spot in the
colorimetric comparison with a sample of the named
chramatogram obtained with solution (2) (2%) and not more
substance, for example, 4-chloroaniline in Chlorhexidine
¡han one such spot is more intense than the spot in the
Irrigation Solution and 4-aminophenol in Benorilate
chromatogram obtained wüh solutian (3) (0.5%).
Tablets.
10.2Where it is known that several impurities are likely to be
9.2 Where a specimen of the impurity is required in the test,
present at significant concentrations, a three-level test
this will be made available as a Chemical Reference
may be appropriate. An example is Pentazocine
Substance unless it is known that specimens of the
Injection:
requisite quality can readily be obtained through the
usual suppliers of chemical reagents. By each method of visualisation, in the chromatogram
abtained with solution (1) any secondary spot is nat more
9.3 Specific control may be inc1uded within a more general
intense than the spot in the chromatagram obtained with
test controlling other impurities; an example is
solutian (2) (1%), not more than one such spot is more
4-epianhydrotetracyc1ine in the monograph for
intense than the spot in the chromatogram obtained with
Lymecyc1ine.
solurian (3) (0.5%) and nat more than four such spors are
9.4 In other cases, an absolute method is more appropriate. more intense than rhe spot in the chromatogram obtained
Such a test may be for a group of potentially toxic wüh salutian (4) (0.25%).
impurities, e.g., polycyclic aroma tic hydrocarbons in
10.3General tests with an 'open' design such as those
Liquid Paraffin.
described aboye have the great advantage that they
9.5 Sometimes an impurity may be named in the provide a means of limiting the levels of related
Pharmacopoeia because it is necessary to use a named substances that may arise from modified or alternative
substance in its control for analytical reasons, such as synthetic routes not in use at the time the test was
different response factors in the specified test method. elaborated. In this context, thin-layer chromatography
Examples are iminodibenzyl in Imipramine Tablets, and
V-A618 Supplementary Chapter 1 A 2014
has the advantage over liquid chromatography and gas In the context of a related substances test where a correction
chromatography that it allows detection of impurities factor is quoted for an impurity, unless otherwise stated, this
completely retained or those not retained at all by the is the expected correction for that impurity in relation to a
stationary phase. response of unity for the substance being examined. The way
11. Total impurity limits In gas chromatographic and in which a correction factor is to be used in any subsequent
liquid chromarographic tests, it is increasingly common ro calculation is stated in the monograph.
limit the total areas of peaks due to related substances. 17. Correction factors of les s than 0.2 or more than 5 are not
In monographs currently in force the limit for the sum is generally used. If the difference between the response of an
commonly in the range 1 to 2% but these values are impurity and that of the substance being examined is outside
applicable at the end of the shelf-life. This procedure is rarely these limits, a different method of determination, such as a
adopted in thin-Iayer chromatographic tests, because of the different detection wavelength (A.) or a different method of
semi-quantitative nature of estimating individual spots and visualisation, is used.
resulting imprecision in expression of results for the totals. 18. For a correction factor quoted in a pharmacopoeial test,
This apparent drawback to the use of thin-Iayer the following points are observed:
chromatography is largely overcome by means of two- and (a) the peak to which the factor applies is identified
three-Ievel tests as described in paragraphs 10.1 and 10.2, unambiguously, recognising the difficulties associated
aboye.
with such identification in chromatograms showing peaks
12. In response ro requests from users of the Pharmacopoeia, of similar retention times (the use of a sample containing
statements of the approximate real or nominallevels of an unquantified amount of the relevant impurity can
impurities controlled by tests for impurities have been widely sometimes be used to assist such identification],
introduced, where appropriate, for information (see (b) the correction factor quoted is a confirmed value and
paragraphs 14 and 15 belowl. Conformity with the preferably is based on relative peak areas of equal
requirements will still be determined on the basis of concentrations of the impurity and the sample under the
compliance or otherwise with the stated test. conditions of the test (altematively the reciprocal of an
Test design and expression oflimits absolute figure based on the A(l %, 1 cm) for the
13. For identified impurities, several aspects are taken into detection wavelength used (or similar for other methods
account in designing the test. These include the nature of the of detection) of the impurity and the sample may be
impurity, its toxicity and the levels likely to be found in usedl,
routine production. Analytical considerations such as the (c) the correction factor is stated in a way that does not
correction factor (defined in paragraph 16) for the imply an unrealistic or misleading degree of precision
impurity and practical issues such as availability of the (declaration to several significant figures is not
impurity as a reference material or reagent also influence the meaningful when placed in context of the nature of the
test designo test and the amount of the impurity likely to be present
14. If a major andlor toxic impurity in a material is known to (typically, less than 0.5%) (e.g., 1.4, 2.5, 3.0, 0.5) or 1
have a significantly different response (more than ± 25%) decimal place (e.g., 1.4,7.5) are gene rally appropriate],
from that of the main peak in the substance being examined (d) older monographs of the BP may contain the term
in the conditions of the test, the preferred manner of limiting response factor. The term will be revised to correction
this impurity is to use a reference substance of the impurity. factor as these monographs are updated.
If this is not possible, a reference solution of the substance 19. Identification of peaks is generally not based on absolute
being examined containing a known amount of the retention times since these may be too 'system dependent';
impurity may be used. Using either of these approaches, the however, advice such as 'the principal peak has a retention
concentration limit iridicated in the monograph for time of about x minutes' may be given. In sorne cases, for
information in parentheses expresses the approximate limit as examp1e, where a simple chromatogram is expected ro show
a real percentage of the impurity in question. This is also a limited number of impurities, ah expected relaúve retention
referred to as % w/w. When neither of these approaches is may be given to designate impurities. In other cases where
possible, a dilution of the solution of the substance being potential impurities have similar retention, asample
examined may be used as a reference solution. This approach containing the components of interest and a sample
is also commonly used in tests where an impurity that is chromatogram may be provided.
known (but not named within the test) has a response within 20. Unidentified impurities may be limited by reference to
± 25% of that of the main peak in the substance being a dilution of the solution of the substance being examined
examined. used as a reference solution together with an open design of
15. Unless explicitly stated otherwise, an indication of the statement limiting 'any' or 'any other' secondary peak or spot.
approximate concentration limit provided in any test where a Such a reference solution may be used in addition to those
dilution of the substance being examined is used as the containing named impurities (any other secondary peak/spot)
reference solution should be interpreted as an express ion in or, in sorne simple tests, control of unspecified and specified
terms of a nominal percentage of the substance being (but unnamed) impurities may be exerted by means of a
examined (in accordance with the General Notices) rather comparison between the sample solution and a dilution of
than as a real percentage of the impurity. this solution (any secondary peak/spot).
16. No reference is made in a test to a correction factor for 21. An indication of the approximate limit concentration for
an identified impurity unless unavoidable. When used, the an unidentified impurity would only be given in terms of a
term is defined as follows: nominal percentage of the substance being examined (see
The correction factor (l/k) is a relative term, being the paragraph 15) since no assumption can be made about the
reciprocal of the peak response of equal concentrations response of an unidentified impurity. While such nominal
of one substance relative to that of another in the limits are not quantitatively fully transparent and caution is
conditions described in the test. needed in anempting to use them to set a total
2014 Supplementary Chapter 1 B V -A61 9
impurity limit, their use is less misleading than quoting an (Supplementary Chapter IV D). At present, a test for solvent
arbitrary/assumed correction factor of 1 for an unidentified residues is included in a specific monograph only where
impurity. variation in levels of known solvents requires control, e.g.,
methanol in Gentamicin Sulfate.
Formulated Preparations 28. The British Pharmacopoeia Commission welcomes
22 . Many monographs for formulated preparations in the suggestions for improving the monographs. In particular,
British Pharmacopoeia and the British Pharmacopoeia where it is found that significant impurities are not controlled
(Veterinary) also include tests for impurities. In general, by the monograph, the Commission would be glad ro receive
wherever possible a test for impurities based on that in the details of validated methods that can be considered for
monograph for the active ingredient is included with any adoption.
necessary modification.
23. Wider limits andlor additional controls may be required
for impurities arising on manufacture or storage of the dosage
form o
B. Polymorphism
l . Polymorphism (the occurrence of more than one morphic
24. Tests for impurities in monographs for formulated
form) is a function of the internal structure of crystalline
preparation are used to control not only degradation
solids. Its occurrence cannot be predicted and, as it can be
products but also by-products of the synthetic route used for
induced in many materials in appropriate conditions, its
manufacture of the active ingredient. It has been argued, for
absence is difficult to demonstrate using a single, specific
example in the ICH guideline Q3B, that by-products of
test. Different polymorphs may exhibit different
synthesis have been controlled already during examination of
physicochemical properties such as melting point, dissolution
the substance before formulation and that fur1her testing for
rate and infrared absorption spectrum. In sorne cases these
these impurities is unnecessary. Clearly it would be
may affect the handling characteristics of the material, the
repetitious and wasteful of resources for tests, often complex
stability of formulated preparations and bioavailability.
in nature, ro be repeated routinely simply to demonstrate
Control of morphic form by a manufacturer is necessary
acceptably low levels of impurities that could arise only
during processing of active ingredients and excipients and
during synthesis (as opposed to degradation) of the active
during production of a formulated product 10 ensure the
ingredient. However, this information is available only to
correct physical characteristics of the product. Its main
those who know the detailed attributes of the active raw
importance to the control of medicinal products is in the
material that has been used. For an analyst who has access
areas of bioavailability and stability.
only ro the dosage form, the profile of synthesis-related
impurities offers one means of establishing whether or not 2. The morphic form of a readily soluble starting material
the dosage form has been prepared from an active ingredient that is incorporated into a solution, for example, an injection,
of pharmacopoeial quality. It is for this reason that such tests an oral solution or eye drops, is not usually important.
are included in British Pharmacopoeia and British (An exception ro this statement might be if the concentration
Pharmacopoeia (Veterinary) monographs for formulated of the solution is such that it is close to the limit of solubility
preparations [see paragraph 3 of General considerations in of one of the possible polymorphs.) The morphic form may
the introduction ro Supplementary Chapter I]. be important when the material is included in a solid dosage
form or as a suspension in a liquid dosage form when the
Current and future deve10pments characteristics of the different polymorphs are such as to
affect the bioavailability of the material.
25. Transparency A statement giving the identities of
impurities that are known to be limited by the specifications 3. Pharmaceutical and medicinal substances For most
is being added to appropriate monographs for medicinal substances it will not usually be appropriate or necessary for
substances and formulated preparations. For example the the monograph 10 control the morphic formo There are,
monograph for Diamorphine Hydrochloride contains the however, a few monographs that restrict the substance to a
following information at the end of the monograph: single formo This may be done by permitting no deviation
from the spectrum/form of the reference substance prescribed
. IMPURITIES The impurity limited by the chis monograph
or by restricting the melting point rimge. An example is .
is: 6-0-acetylmorphone
Carbamazepine where the infrared spectrum is determined
This increase in the transparency of pharmacopoeial without prior treatrnent of the sample and the melting range
specifications is of assistance to licensing authorities and permitted excludes the polymorph of lower melting point.
others when considering whether the standards in the
4. Where it is known that the substance exists in more than
monograph are appropriate ro a new source of supply. It is
one morphic form a statement is frequently included in the
emphasised that other, unnamed impurities may also be
monographs of the Pharmacopoeia under Characteristics, for
limited. ihe Commission is actively seeking information that
example, the monographs for Dextropropoxyphene Napsilate
will allow the statements ro be extended in future.
and Spironolactone. In the absence of such a statement, the
26. System suitability In chromatographic tests increasing existence of polymorphism may sometimes be deduced from
use is being made of system suitability tests to enable the the tests of the monograph. The most common example is
analyst 10 confirm that performance of the chosen column or found in infrared identification tests where the analyst may
plate is satisfac10ry under the chosen conditíons. In liquid be instructed 10 prepare a solution spectrum or recrystallise
chromatographic and gas chromarographic tests, peak the sample if the spectrum obtained is not concordant with
separatíon between impurities and the substance being the reference spectrum or spectrum of a reference substance,
examined is generally considered to offer the best indication for example, Prednisolone Sodium Phosphate. This
of performance of the system. procedure recognises polymorphism but does not limit the
27. Residual solvents A general test for residual solvents form permitted.
has been included in the European Pharmacopoeia 5. It is intended to extend the inclusion of explicit statements
(Appendix VIII L) together with guidelines on its application in monographs for pharmaceutical and medicinal substances
V-A620 Supplementary Chapter 1 C 2014
as information on the occurrence of polymorphism becomes in the United States of America (Ee6). Following adoption
available. The eommission would we1come information from of the recommendations in the report of the collaborative
users of the Pharmacopoeia in order that such statements can studyI global harmonisation of endotoxin unitage has been
be added in appropriate cases. achieved, that is, the FDAlUSP Endotoxin Unit (EU) is
6. Formulated preparations Polymorphism is a potential equivalent to the Intemational Unit (IU).
problem in solid dosage forms, such as tablets, or in liquid or
General policy
semi-solid preparations where the active ingredient is present
as a solid, for example, in oral suspensions. In most cases it 4. In any individual monograph only one test is required,
is difficult to demonstrate that the material contains the either that for pyrogens or that for bacterial endotoxins .
desired polymorph as the process of extracting a sufficient 5. In the absence of evidence to the contrary, the test for
sample of the .material in question for analysis may itse1f bacterial endo1Oxins is preferred, since it is considered usually
change the form of the material. For a solid dosage form to provide equal or better protection to the patient.
where the active ingredient is known to exist in forms with 6. Before inc1uding a test for bacterial endotoxins in a
significant differences in solubility, a dissolution test may be monograph, evidence is required that a test, as described in
the method of choice. Appendix XN e , can be applied satisfactorily 10 the item in
7. Where the active ingredient is known to exist in more than question.
one morphic form and the choice of polymorph is critical 7. The necessary information is sought from manufacturers.
with regard to bioavailability ami/or stability, the method of eompanies are invited to provide any validation data that
manufacture should ensure the presence of the correct they have conceming the applicability of the test for bacterial
amount of the desired polymorph in the preparation. endo1Oxins 10 the substances and formulations of interest.
In future the side heading 'Production' will be used to draw 7.1 Such data should inc1ude details of sample preparation
attention to control of morphic form during manufacture in and of any procedures necessary 10 elimina te interfering
cases where control of morphic form is known to be fac1Ors.
important.
7.2 In addition, any available paralle1 data for rabbit pyrogen
8. Such Production statements might also inc1ude reference testing that would contribute to an assurance that the
to the need during product development to examine drug replacement of a rabbit pyrogen test by the test for
sensitivity to polymorphic change due to granulation bacterial endo1Oxin is appropriate, should be provided.
conditions or compressional force s and to make appropriate
7.3 For formulated preparations, a distinction should be
processing adjustments to control potential variation.
made between any sample treatment necessitated by
9. The eommission would find it helpful to be advised of excipients inc1uded in a particular product and any
instances where control of morphic form is important so that required due to the nature of the active constituent.
a suitable Production statement may be added. In such cases Attention should also be drawn 10 any manipulation
it would also appreciate receiving details of any validated test normally required for the active constituent that is
methods that will appropriately limit the undesirable formo rendered redundant by the composition of the
formulation, for example, pH adjustment.
8. In order to set an appropriate limit for bacterial
endotoxins it is necessary 10 know the intended route of
C. Bacterial Endotoxin Testing parenteral administration (in particular, if the substance may
This section pro vides an exposition of the Commission 's policy and be administered intrathecally) together with the maximum
information on its implementation. The guidelines of the European dose as recommended in relevant product data sheets.
Pharmacopoeia are included as an Annex. The limit for a given material or preparation is expressed as
The test for bacterial endotoxins of the European the endo1Oxin limit (EL) or endotoxin limit concentration
Pharmacopoeia (Ph Eur) is inc1uded as Appendix XN e of (EL). The EL may be expressed in IU of endotoxin per -
the British Pharmacopoeia. This text has been prepared in millilitre for a defined solution of the material or preparation,
collaboration with the Japanese Pharmacopoeia and the or, for sorne medicinal substances, in IV of endotoxin in
United States Pharmacopeia. relation to a defined quantity of the material (that is, per
1. This in vitro test is being progressively applied in milligram or, for biologically assayed materials, per IV) of
appropriate monographs of both the British and European material. The limit, which is stated in the monograph, is
Pharmacopoeia in place of the in vivo test for pyrogens . usually established on the following basis:
2. Methods for the detection of Gram-negative bacterial EL = K/M
endotoxins are based on the use of a lysate of amoebocytes 8.1 K, sometimes referred to as the minimum pyrogenic
from the horseshoe crab (Limulus polyphemus or Tachypleus dose, is the maximum number of IU of endotoxin which
tádentatus). Addition of endotoxin to this lysate may result in the patient may receive without suffering toxic reactions.
gelation, precipitation or turbidity. The method used in the The appropriate value for K will be taken from the table
monographs of the European and British Pharmacopoeias is, below.
unless otherwise stated, that using a gelation end-point. 8.2 Mis the maximum dose of the drug substance per
3. A European Pharmacopoeia Biological Reference person (or per kg) per hour. This is interpreted as the
Preparation (BRP) of Endotoxin calibrated in Intemational maximum amount that might be administered within
Units (IU) has beenestablished for use in this test. one hour. For subcutaneous (Se), intramuscular (1M)
The current BRP (batch 4) was established following an or bolus intravenous (N) injections this will be an entire
intemational collaborative study. It consists of endotoxin single dose. For intravenous infusions given over a
from the same bulk as the Second Intemational Standard
established by the World Health Organization and the J Poole, s., Dawson, P , Gaines Das, R.E. (1997). Second internarional
current standard established by the Food and Drug standard for endotoxin: calibrarion in an international co/laborative study.
Administration and the United States Pharmacopeia for use Journal ofEndotoxin Research 4 (3), 221 -23 1.
2014 Supplementary Chapter 1 C V-A621
Type of product Route of administration K

per person perkg
A11 parenteral preparations Intrathecal l 14 0.2
Radiopharmaceuticals Intravenous 175 2.5
AIl parenteral preparations except AIl parenteral routes except 350 5.0
intrathecal radiopharmaceuticals
lWhere a product can be adminisrered both intrathecally and by another parenteraJ rOUle, the more stringent
value of K for the intrathecal mute will be raken as che basis of establishing the ELC.
prolonged period it is the proportion of the dose that other pyrogenic substances. These endolOxins are
would be infused during one hour and depends upon lipo-polysaccharides. Although there are a small number of
the rate of infusion. The value used is the maximum pyrogens which possess a different structure, the conclusion
dose recommended by the manufacturer and stated in is generally justified that the absence of bacterial endotoxins
the relevant product data sheet. It is accepted that in in a product implies the absence of pyrogenic components,
exceptional circumstances this dose may be exceeded at provided the presence of non-endotoxin pyrogenic substances
the discretion of the physician; such use is outside the can be ruled out.
scope of the Pharmacopoeia. The presence of endolOxins in a product may be masked by
factors interfering with the reaction between the endolOxins
Implementation
and the amoebocyte Iysate. Hence, the analyst who wishes lO
9. The British Pharmacopoeia Commission is seeking to replace the rabbit pyrogen test required in a pharmacopoeial
replace the test for pyrogens by that for bacterial endolOxins monograph by a test for bacterial endolOxins has lO
wherever possible in the Pharmacopoeia. The European demonstrate that a valid test can be carried out on the
Pharmacopoeia Comrnission has a similar policy and has product con cerned; this may entail a procedure for removing
indicated that, for monographs already published, the change interfering faclOrs.
will be carried out, where appropriate, whenever the
As indicated in the test for bacterial endolOxins (2.6.14),
monograph in question is revised.
information must be available on the 2 following aspects
9.1 The test for bacterial endotoxins is now specified in the before a test on a sample can be regarded as valid.
European Pharmacopoeia monograph for Water for
- The suitability of the material lO be used for the test has
Injections and in many other monographs including a
lO be established. The absence of endolOxins in the water
range of antibiotics (for example, Doxorubicin
for BET and in the other reagents must be assured and
Hydrochloride and Oxytetracycline Hydrochloride),
the sensitivity of the amoebocyte Iysate must be checked
biological materials (for example, Somatropin and
to confirm the sensitivity declared by the manufacturer.
Heparin) and radiopharmaceutical preparations.
- As the product to be examined may interfere with the test,
9.2 Revision of the European Pharmacopoeia monograph for
the sensitivity of the amoebocyte Iysate is determined in
Parenteral Preparations lO permit the use of the test for
the presence and in the absence of the product under
bacterial endolOxins in defined circumstances opened the
examination. There must be no significant difference
way for the test for pyrogens to be replaced by that for
between the 2 sensitivity values.
bacterial endolOxins in individual monographs for
parenteral preparations in the British Pharmacopoeia. The text 2.6.14. Bacterial endotoxins indica tes methods for
The necessary change has already been made in a wide removing interfering faclOrs; in the case of interference,
range of monographs including those for certain another test must be carried out after such a method has
biological formulations such as Protamine Sulfate been applied to check whether the interference has indeed
Injection and in the monographs for a number of widely been neutralised or removed.
used intravenous infusions such as Sodium Chloride This general chapter explains the reasons for the
Intravenous Infusion. Appropriate data are being sought requirements in the test for bacterial endotoxins, then deals
from manufacturers for the remaining monographs for with the reading and interpretation of the results .
which a test for pyrogens is specified. Substitution of the rabbit pyrogen test required in a
pharmacopoeial monograph by an amoebocyte Iysate test
Annex
constitutes the use of an alternative method of analysis and
Guidelines conceming the test for Bacterial endotoxins have been hence requires validation; sorne guidance on how lO proceed
pul:;lished as an Annex to the method text 2.6.14 in the European is given in section 11.
Pharmacopoeia and are reproduced here. The reference method for bacterial endolOxins is stated in the
The following seaion is published for information. monograph on a given product; where no method is stated,
method A is the reference method. If a method other than
Guidelines For Using The Test For Bacteria!
the reference method is lO be used, the analyst must
Endotoxins
demonstrate that the method is appropriate for this product
(Ph. Eur. method 5. 1. 10) and gives a result consistent with that obtained with the
1. Introduction reference method (see also Section 13).
Endotoxins from gram-negative bacteria are the most 2. Method
common cause of toxic reactions resulting from
The addition of endolOxins lO amoebocyte Iysate may result
contamination of pharmaceutical products with pyrogens;
in turbidity, precipitation or gelation (gel-clot); only the
their pyrogenic activity is much higher than that of most
V-A622 Supplememary Chapter 1 C 2014
gel-clot method was used in the Pharmacopoeia as an The endotoxin limit depends on the product and its route of
evaluation cl'iterion in the first type of test fol' bacterial administration and is stated in the monograph. Values for K
endotoxins. The advantage was the simplicity of basing the are suggested in Table 5.1.10.-1.
decision to pass or fail the product undel' examination on the For other routes, the acceptance criterion for bacterial
absence 0 1' presence of a gel-clot, visible with the naked eye. endotoxins is generally determined on the basis of results
The quantitative methods described as methods C, D, E and obtained during the development of the preparation.
F were developed later: they l'equire more instrumentation,
but they are easier to automate fol' the regular testing of lal'ge
Table 5.1.10.-1
numbers of samples of the same product.
Route oC administration K (IV oC endotoxin per kHogram oC body mass)
Endotoxins may be adsorbed onto the surface of tubes or
pipettes made from certain plastics or types of glass. Intravenous 5.0
Interference may appear due to the release of substances Intravenous. for 2.5
from plastic materials. H ence, the materials used should be radiopharmaceu ticals
checkedj subsequent batches of tubes or pipettes may have a Intrathecal 0.2
slightly different composition, and therefore the analyst is
advised to repeat such tests on starting with new batches of
materials. Which dilution of the product is to be used in the test to
obtain maximal assurance that a negative result means that
The decision to use the test for bacterial endotoxins as a
the endotoxin concentration of the product is less than the
limit test implies first that a threshold endotoxin
endotoxin limit and that a positive resuIt means that the
concentration must be defined for the product to be tested,
lysate detected an endotoxin concentration equal to or
and second that the objective of the test is to know whether
greater than the endotoxin limit? This dilution depends on
the endotoxin concentration in the product under
the endotoxin limit and on the sensitivity of the lysate: it is
examination is below or abo ve this threshold.
called the M aximum Valid Dilution (MVD) and its value
The quantitative methods C, D, E and F make it possible to
may be calculated using the following expression:
determine the endotoxin concentration in the sample under
examination, but for compliance with the Pharmacopoeia and
endo toxin limit x concentr a tion of test solution
in routine quality control the final question is whether or not
A
this concentration exceeds a defined limit.
In setting a threshold concentration of endotoxin for the
product to be tested, due attention should be paid to the Concentration 01 test solution:
dos e of the product: the threshold should be set so as to - mg/mL if the endotoxin limit is specified by mas s
ensure that as long as the endotoxin concentration in the (IU/mg),
product remains below this threshold even the maximal dose - Units/mL if the endotoxin limit is specified by unit of
administered by theintended route per hour does not biological activity (IU/Unit),
contain sufficient endotoxin to cause a toxic reaction. - mUmL if the endotoxin limit is specified by volume
When the endotoxin concentration in the product exactly (IU/mL).
equals the threshold value, gelation will occur, as is the case
), = the labelled lysate sensitivity in the gel-clot technique
when the endotoxin concentration is much higher, and the
(IU/mL) or the lowest concentration used in the
product will fail the test, because the all-or-none character of
standard curve of the turbidimetric or chromogenic
the test makes it impossible to diffe rentiate between a
techniques.
concentration exactly equal to the threshold concentration
and one that is higher. Ir is only when no gelation occurs When the value of the maximum valid dilution is not a whole
that the analyst may conclude that the endotoxin number, a convenient whole number smaller than the MVD
concentration is below the thl'eshold concentration. may be used for routine purposes (which means preparing a
For products in the solid sta te, this threshold concentration solution of the product which is les s diluted than the MVD
of endotoxin per mass unit or per Intemational Unit (IU) of indicates). In this case, a negative result indicates that the
product has to be translated into a concentration of endotoxin concentration of the product lies below the limit
endotoxin pel' millilitre of solution to be tested, as the test value. However, when the endotoxin concentration of the
can only be carried out on a solution. The case of produ cts product in such a test is les s than the endotoxin limit but
that already exist in the liquid state (such as infusion ftuids) high enough to make the reaction with the lysate resuIt in a
is discussed below. clot, the test may be positive under these conditions. Hence,
Endotoxin [imit the endotoxin limit for active substances when a test with this 'convenient' dilution factor is positive,
the product should be diluted to the MVD and the test
administered parenterally, defined on the basis of dose, is
equal to: should be repeated. In any case of doubt or dispute the
MVD must be used.
K This stresses the importance of the confirmation of the
M sensitivity of the lysate.
EXAMPLE
K threshold pyrogenic dose of endotoxin per kilogram of A 50 mg/mL solution of phenytoin sodium (intended for
body mass, intravenous injection) has to be tested. Determine the MVD,
M maximum recornmended bolus dose of product per given the following variables:
When the product is to be injected at frequent intervals or
infused continuously, M is the maximum total dose
administered in a single hour periodo
2014 Supplementary Chapter 1 C V-A623
M maximum human dose = 15 mg per kilogram of body 7. Preliminary test for interfering factors
mass, Sorne products cannot be tested directly for the presence of
e 50 mg/mL, endotoxins because they are not miscible with the reagents,
K 5 IU of endotoxin per kilogram of body mass, they cannot be adjusted to pH 6.0-8.0 or they inhibit or
le 0.4 IU of endotoxin per millilitre. activate gel formation . Therefore a preliminary test is
required to check for the presence of interfering factors;
when these are found the analyst must demonstrate that the
MVD = 5 x 50 x _1_ = 41.67 pro ce dure to remove them has been effective.
15 0.4
The object of the preliminary test is to test the null
For routine tests on this product, it may be expedient to hypothesis that the sensitivity of the Iysate in the presence of
dilute 1 mL of the solution to be tested to 20 mL (MVD/2 the product under examination does not differ significantly
rounded to the next lower whole number). However, if this from the sensitivity of the lysate in the absence of the
test result is positive the analyst will have to dilute 1 mL to product. A simple criterion is used in methods A and B: the
41.67 mL and repeat the test. A dilution to 41.67 mL is also null hypothesis is accepted when the sensitivity of the Iysate
necessary when the test is performed to settle a dispute . in the presence of the product is at least 0.5 times and not
more than twice the sensitivity of the lysate by itself.
3. Reference material A classical approach would have been to calculate the means
Endotoxin standard BRP is intended for use as the reference of the log dilution factor for the Iysate sensitivity with and
preparation. It has been assayed against the WHO without the product and to test the difference between the 2
Intemational Standard for Endotoxin and its potency is means with Student's t-test.
expressed in Intemational Units of endotoxin per ampoule. The test for interfering factors in gel-clot methods A and B
The Intemational Unit of endotoxin is defined as the specific requires the use of a sample of the product in which no
activity of a defined mass of the Intemational Standard. endotoxins are detectable. This presents a theoretical
For routine purposes, another preparation of endotoxin may problem when an entirely new product has to be tested.
be used, provided it has been assayed against the Hence, a different approach was designed for quantitative
Intemational Standard for Endotoxin or the BRP and its methods C, D, E and F.
potency is expressed in Intemational Units of endotoxin.
8. Removal of interfering factors
NOTE 1 International Unit (IU) 01 endotoxin is equal to 1
The procedures to remove interfering factors must not
Endotoxin Unit (E. U.).
increase or decrease (for example, by adsorption) the amount
4. Water for bet of endotoxin in the product under examination. T he correct
way of checking this is to apply the procedures to a spiked
Testing the absence of endotoxin in this reagent by a
sample of the product, that is, a sample to which a lznown
technique derived from rhe rabbit pyrogen test was rejected
for practical and th'eoretical reasons: amount of endotoxin has been added, and then to measure
the recovery of the endotoxin.
- the rabbit test is not sensitive enough to detect endotoxin
in water for BET intended for tests on products with a
Methods e and D If the nature of the product to be
analysed shows interference which cannot be removed by
very low endotoxin limit;
classical methods, it may be possible to determine the
- the relatively low precision of the rising temperature standard curve in the same type of product freed from
response in rabbits would call for many replications in endotoxins by appropriate treatment or by dilution of the
rabbits; product. The endotoxins test is then carried out by
- the terms 'pyrogens' and 'endotoxins' denote groups of comparison with this standard curve.
entities that do not coincide completely. Ultrafiltration with cellulose triacetate asymmetric membrane
The text 2.6.14. Bacterial endotoxins indicates that methods filters has been found to be suitable in most cases. The filters
other than triple distillation may be used to prepare water for should be properly validated, because under sorne
BET. Reverse osmosis has been used with good results; sorne circumstances cellulose derivatives W-d-glucans) can cause
analysts may prefer to distil the water more than 3 times . false positive results.
Whatever method is used, the resultant product must be free Polysulfone filters have been found to be unsuitable because
of detectable endotoxins . false positive results had been obtained by sorne users.
5. pH ofthe mixture 9. The purpose ofthe controls
In the test for bacterial endotoxins, optimum gel-clot occurs The purpose of the control made up with water for BET and
for a mixture at pH 6.0-8.0. However, the addition of the the reference preparation of endotoxin at twice the
lysate to the sample may result in a lowering of the pH. concentration of the labelled lysate sensitivity is to verify the
activity of the lysate at the time and under the conditions of
6. Validation of the lysate
the test. The purpose of the negative control is to verify the
It is important to follow the manufacturer's instructions for absence of a detectable concentration of endotoxin in water
the preparation of the solutions of the lysaté. for BET.
The positive end-point dilution factors in gel-clot methods A The positive control, which contains the product to b e
and B are converted to logarithms. The reason is that if the examined at the concentration used in the test, is intended to
frequency distribution of these logarithmic values is plotted, show the absence of inhibiting fa ctors at the time and under
it usually approaches a normal distribution curve much more the conditions of the test.
closely than the frequency distribution of the dilution factors
themselves; in fact it is so similar that it is acceptable to use 10. Reading and interpretation ofthe results
the normal frequency distribution as a mathematical model Minute amounts of endotoxin in the water for BET, or in
and to calcula te confidence limits with Student's t-test. any other reagent or material to which the lysate is exposed
V-A624 Supplementary Chapter 1 D 2014
during the test, may escape detection as long as they do not compliance with the standards of the monographs would
reach the sensitivity limit of the lysate. However, they may be achieved if the official methods were used. In the
raise the amount of endotoxin in the solution containing the event of doubt or dispute, the methods of analysis of the
product under examination to just aboye the sensitivity limit Pharmacopoeia are alone authoritative."
and cause a positive reaction. The following procedures are suggested for validating a
The risk of this happening may be reduced by testing the method for bacterial endotoxins other than the one implied
water for BET and the other reagents and materials with the or indicated in the monograph.
most sensitive lysate available, or at least one that is more 13.1The procedure and the material s and reagents used in
sensitive than the one used in the test on the product. Even the method should be validated as described for the test
then, the risk of such a 'false positive result' cannot be ruled concerned.
out completely. It should be realised, however, that in this 13.2The presence of interfering factors (and, if needed, the
respect the test design is 'fail-safe' in contrast to a test design procedure for removing them) should be tested on
permitting a false negative result, which could lead to the samples of at least 3 production batches. It should be
release of an unsatisfactory product, thus endangering the borne in mind that methods D and E, using a
patient's health. chromogenic peptide, require reagents that are absent in
11. Replacernent ofthe rabbit pyrogen test by a test methods A, B, C and F, and hence compliance of
for bacterial endotoxins methods A, B, C or F with the requirements for
interfering factors cannot be extrapolated to method D
Monographs on pharmaceutical products intended for
or method E without further testing.
parenteral administration that may contain toxic amounts of
bacterial endotoxins require either a test for bacterial 14. Validation ofthe testfor new products
endotoxins or a rabbit pyrogen test. As a general policy: The procedures described under 13-1 and 13-2 should be
- in any individual' monograph, when a test is required, only applied to all new products intended for parenteral
one test is ineluded, either that for pyrogens or that for administration that have to be tested for the presence of
bacterial endotoxins; bacterial endotoxins according to the requirements of the
- in the absence of evidence to the contrary, the test for Pharmacopoeia.
bacterial endotoxins is preferred over the test for
pyrogens, since it is usually considered to provide equal or
better protection to the patient; D. Excipients
- before ineluding a test for bacterial endotoxins in a l. The General Notice on Excipients states that 'any
monograph, evidence is required that one of the tests substances added in preparing an official preparation shall ...
described in chapter 2.6.14 can be applied satisfactorily to not interfere with the assays and tests of the Pharmacopoeia'.
the product in q1,lestion; 2. The British Pharmacopoeia Commission wishes to stress
- the necessary information is sought from manufacturers; that any preparation described by a name at the head of a
companies are invited to provide any validation data that monograph in the current edition of the Pharmacopoeia,
they have concerning the applicability of the test for whether or not it is referred to as BP, is not of pharrnacopoeial
bacterial endotoxins to the substances and formulations of quality unless it meets all of the requirements of che
interest; such data ineludes details of sample preparation monograph when tested by the methods set down.
and of any procedures necessary to eliminate interfering 3. It is recognised that new formulations of existing
factors; in addition, any available parallel data for rabbit preparations may from time to time be developed and that
pyrogen testing that would contribute to an assurance that the excipients and other ingredients used might result in
the replacement of a rabbit pyrogen test by the test for interference with the official assays and tests. In such cases
bacterial endotoxin is appropriate, must be provided. the Commission is prepared to consider modification of
Additional requirements are defined in the following sections. methods to overcome the difficulties thus caused.
4. When seeking modification manufacturers are invited to
12. Use of adifferent bacterial endotoxin test from submit details of the nature of the interference together with
that prescribed in the monograph proposals for change that will allow the valid testing, by an
When a test for bacterial endotoxins is prescribed in a independent analyst, not only of the proposed new
monograph and none of the 6 methods (A to F) described in formulation but also of al! similar preparations already on the
chapter 2.6.14 is specified, then method A, the gel-elot market. Practical evaluation in the Laboratory will usually be
method limit test, has been validated for this producto If one necessary before any amendments to a British
of the other methods (B to F) is specified, this is the one Pharrnacopoeia monograph can be considered. It is therefore
which has been validated for this product. in the interests of a manufacturer to submit any proposals at
the earliest possible date.
13. Validation of alternative methods
Replacement of a rabbit pyrogen test by a bacterial endotoxin
test, or replacement of a stated or implied method for E. Dissolution Testing of Solid Oral
bacterial endotoxins by another method, is to be regarded as
the use of an alternative method in the replacement of a Dosage Forms
pharmacopoeial test, as described in the General Notices: This section provides information on the phannacopoeial
"The test and assays described are the official methods dissolution test and guidance on its function and application in
upon which the standards of the Pharmacopoeia are individual monographs of the British Pharmacopoeia for tablets
based. With the agreement of the competent authority, and capsules.
alternative methods of analysis may be used for control 1. Harmonisation
purposes, provided that the methods used enable an 1.1 The dissolution test for solid oral dosage forms in
unequivocal decision to be made as to whether Appendix XII Blof the British Pharmacopoeia is that of
2014 Supplementary Chapter 1 E V-A625
Is a dissolution test specified

in the individual monograph?
Yes I No
~
Is a dissolution test indicated in the
dosage form general monograph?
Yes No
¡ ¡
Perform the dissolution test Unless otherwise justified Dissolution testing
using the method specified and authorised, perform the is not required.
in the monograph . dissolution test using a
validated in-house method.
~ ~
Is a limit specified in the No Was the monograph first
test? published before SP 2008?
Yes Yes I No
¡ ¡
Apply the limit Apply the established Apply the harmonised
specified in the SP criteria (not less criteria (Q = 75% of
monograph than 70% of label label claim in 45 min)
claim in 45 min)
Figure SCIE-l
the European Phannacopoeia (Ph. Eur. method 2.9.3). cell. The descriptions are concordant with those
It is a harmonised text prepared by the Phannacopoeial published in the United States Pharmacopeia (USP).
Discussion Group (PDG). The PDG is comprised of 2.2 Of the two established apparatuses (basket and paddle)
representatives of the European Phannacopoeia, the paddle is now the apparatus of choice for many
Japanese Phannacopoeia and the United States preparations. However, where a published test uses the
Phannacop'eia. Although the test is largely hannonised, basket, work to validate a change to the paddle method
sorne regional differences remain. is not contemplated. The reciprocating cylinder is useful
1.2 It is not the intention of the British Phannacopoeia to for pH profiling studies while the ftow-through cell may
apply retrospectively the hannonised test conditions and be appropriate for preparations of poorly soluble active
acceptance criteria to BP monographs or to change ingredients (see Annex).
unilaterally specifications for existing products . 3. Test eonditions and aeeeptanee eriteria
Appendix XII B 1 contains a section entitled Monographs
3.1 Test conditions The harmonised test conditions
01 the British Pharmacopoeia which provides requirements
included in Appendix XII B1 will be applied to all new
for monographs for tablets and capsules of the BP
monographs of the British Phannacopoeia. It is not the
published prior to 2008. Taking account of pennissible
intention of the British Phannacopoeia Commission to
assay ranges and content unifonnity, this
apply these criteria retrospectively to existing
phannacopoeial (that is, shelf-life) dissolution
monographs. Where an individual monograph prescribes
requirement is considered to offer an acceptab1e degree
the use of the requirements stated under Monographs 01
of assurance of 'complete dissolution' . The choice of a
the British Pharmacopoeia in Appendix XII BI, the
time is, of necessity, somewhat arbitrary but 45 minutes
following conditions using the basket or paddle
is considered satisfactory for the majority of
apparatus are preferred.
conventional-release (non-modified-release) products.
- rotation speed:IOO rpm (basket), 50 rpm (paddle)
2. Apparatus
- dissolution medium volume: 900 mL
2.1 Four types of apparatuses are now described in the
- dissolution medium composition: aqueous, commonly
British and European Phannacopoeias; the basket, the
paddle, the reciprocating cylinder and the ftow-through 0.1 M hydrochloric acid or phosphate buffers of
pH 6.8 to 7.6
V-A626 Supplementary Chapter 1 E 2014
- number of units tested: 6 (plus 6, if a retest is required). Chewable Calciurn Calcium Folinate Tablets
The number of units tested is specified in Carbonate Tablets
Appendix XII BI; other conditions are specified in the
Calcium Gluconate Tablets Effervescent Calcium
relevant individual monographs .
Gluconate Tablets
In situations where it has been demonstrated that the
harmonised criteria are not applicable (e.g. low solubility Calciurn Lactate Tablets Captopril Tablets
preparations, 'coning' of material in the vessel, low Carbamazepine Tablets Carbimazole Tablets
concentration of analyte), modifications may be made to
Cascara Tablets Cefaclor Capsules
the test conditions, such as, adding a surfactant,
increasing the paddle rotation speed or using a modified Cefadroxil Capsules Cefalexin Capsules
vessel and reducing the volume of dissolution medium Cefalexin Tablets Cefradine Capsules
used.
3.2 Acceptance criteria For new monographs for Cefuroxirne Axetil Tablets Chlorambucil Tablets
conventional-release preparations, unless otherwise Chloramphenicol Capsules Chlordiazeooxide Caosules
stated, the harmonised acceptance criteria ("Q" values)
Chlordiazepoxide Hydroch1- Ch1oroquine Phosphate
in Appendix XII BI should be applied. For monographs
oride Tablets Tablets
published prior to the BP 2008, the established BP
criteria using either the basket or the paddle apparatus Chloroquine Sulfate Tablets Ch10rphenamine Tablets
are specified under 'Monographs of the British Chlorpromazine Tablets Chlorpropamide Tablets
PharmacopQeia' in Appendix XII Bl.
Chlortalidone Tablets Choline Theophyl1inate
3.3 The schematic diagram outlines which limits to apply in
Tablets
each case (Fig. SCIE-I).
3.4 A list of those British Pharmacopoeia monographs that Cimetidine Tablets Ciprofloxacin Tablets
were published up to and including the BP 2007 is Clemastine Tablets Clindamycin Caosules
provided below.
Clobazam Capsules Clofazimine Capsules
Acebutolol Capsules Acebutolol Tablets Clofibrate Capsules Clomethiazole Capsules
Acenocoumarol Tablets Acetazolamide Tablets Clomifene Tablets Clomioramine Caosules
Aciclovir Tablets Dispersible Aciclovir Tablets Clonidine Tablets Co-amilofruse Tablets
Alimemazine Tablets Allopurinol Tablets Co-amilozide Tablets Co-amoxiclav Tablets
Aloxiprin Tablets Aluminium Hydroxide Co-beneldopa Capsules Dispersible Co-beneldopa
Tablets Tablets
Alverine Capsules Amantadine Capsules Co-careldopa Tablets Co-codamol Tablets
Amiloride Tablets Aminoglutethimide Tablets Effervescent Co-codamol Co-codaprin Tablets
Tablets
Aminophylline Tablets Amiodarone Tablets
Dispersible Co-codaprin Co-danthrusate Capsules
Amitriptyline Tablets Amoxicillin Capsules
Tablets
Ampicillin Capsules Ascorbic Acid Tablets
Codeine Phosphate Tablets Codergocrine Tablets
Aspirin Tablets Dispersible Aspirin Tablets
Co-dydramol Tablets Co-fluampicil Capsules
Effervescent Soluble Aspirin Gastro-resistant Aspirin
Colchicine Tablets Colecalciferol Tablets
Tablets Tablets
Colistin Tablets Co-magaldrox Tablets
Aspirin and Caffeine Atenolol Tablets
Tablets Co-proxamol Tablets Cortisone Tablets
Atropine Tablets Azapropazone Capsules Co-tenidone Tablets Co-triamterzide Tablets
Azapropazone Tablets Azathioprine Tablets Co-trirnoxazole Tablets Dispersible Co-trimoxazole
Tablets
Baclofen Tablets Bendroflumethiazide Tablets
Paediatric Co-trimoxazole Cyanocobalamin Tablets
Benorilate Tablets Benzatropine Tablets
Tablets
Betahistine Dihydrochloride Betamethasone Tablets Cyclopenthiazide Tablets
Cyclizine Tablets
Tablets
Cyclophosphamide Tablets Cyproheptadine Tablets
Betamethasone Sodium Gastro-resistant Bisacodyl
Phosphate Tablets Tablets Cyproterone Tablets Dapsone Tablets
Bromocriptine Capsules Bromocriptine Tablets Demeclocycline Capsules Desipramine Tablets
Brompheniramine Tablets Bumetanide Tablets Dexamethasone Tablets Dexarnfetamine Tablets
Busulfan Tablets Calcitriol Capsules Dextromoramide Tablets Dextropropoxyphene Caps-
ules
Calcium and Colecalciferol Calcium and Ergocalciferol
Tablets Tablets Diazepam Tablets Diazoxide Tablets
Dichlorophen Tablets Dicycloverine Tablets Hydralazine Tablets Hydroehlorothiazide Tablets

Diethvlcarbamazine Tablets Dieth~ stilb estrol T ablets Hydroeortisone Oromueosal Hydroflumethiazide Tablets
Diflunisal Tablets Digitoxin Tablets Tablets
Digoxin T ablets Hydrotalcite Tablets Hydroxyearbamide C~sules

Dihydrocodeine Tablets
Hydroxyehloroquine Tablets Hyoseine Butylbromide
Diloxanide Tablets Dimen~drinat e Tablets
Tablets
Dipipanone and Cyclizine Dipyridamole Tablets
Tablets Hyoseine Tablets Ibt11'rofen Tablets
Disopyramide Capsules Imipramine Tablets Indapamide Tablets

Disopyramide Phosphate
Capsules Indometaein Capsules Indoramin Tablets
Disulfiram Tablets D ocusate Capsules Inositol Nieotinate T ablets Iopanoie Aeid T ablets
Domperidone Tablets Dosulepin Capsules Isoniazid T ablets Isosorbide Dinitrate Tablets
Dosulepin Tablets Doxepin C:lQsules Isosorbide Dinitrate Isosorbide Mononitrate
Doxycycline Capsules Dispersible Doxycycline Sublingual Tablets Tablets
Tablets Isotretinoin Capsules Isradipine Tablets
Droperidol Tablets Dydrogesterone Tablets Ketoprofen Capsules Labetalol Tablets
Enalapril Tablets Ephedrine Hydrochloride Laeidipine Tablets Levod~a C~ule s
Tablets Levodopa T ablets Levomepromazine T ablets
Ergocalciferol Tablets Ergometrine T ablets
Levonorgestrel Tablets Levonorgestrel and
Ergotamine Sublingual Erythromycin Estolate Ethinylestradiol Tablets
Tablets Capsules Levothyroxine Tablets Lineomyein Capsules
Erythromycin Ethyl Erythromycin Stearate
Liothyronine Tablets Lisinopril Tablets
Succinate Tablets Tablets
Lithium Carbonate Tablets Lofepramine T ablets
Gastro-resistant Estradiol and
Erythromycin Tablets Norethisterone Tablets Lomustine Capsules Loperamide Capsules
Estradiol and Estramustine Phosphate Loprazolam Tablets Lorazepam Tablets
Norethisterone Acetate Capsules Lormetazepam T ablets Lyme9'cline C~sules
Tablets
Compound Magnesium Mebeverine Tablets
Estropipate Tablets Ethambutol Tablets Trisilieate Tablets
Ethinylestradiol Tablets Ethosuximide C~sules Mefenamie Aeid Capsules Mefenamie Acid Tablets
Etodolac Capsules Etodolac Tablets Megestrol T ablets Meloxicam Tablets
Etoposide Capsules Famotidine Tablets Melphalan Tablets Menadiol Phosphate Tablets
Fenbufen Capsules Fenbufen Tablets Meptazinol Tablets Mepyramine T ablets
Fenoprofen Tablets Ferrous Fumarate C~sules Mercaptopurine Tablets Metformin Tablets
Ferrous Fumarate Tablets Ferrous Fumarate and Folic Methadone Tablets Methotrexate Tablets
Acid Tablets
Methylcellulose Tablets Methyldopa Tablets
Ferrous Gluconate Tablets Ferrous Sulfate T ablets
Methylphenobarbital Methylprednisolone Tablets
Finasteride T ablets Flavoxate T ablets Tablets
Fleeainide Tablets Flucloxaeillin Capsules Methysergide Tablets Metoclopramide Tablets
Flueytosine Tablets Fludroeortisone Tablets Metoprolol Tartrate Tablets Metronidazole Tablets
Fluoxetine Capsules Fluphenazine Tablets Metyrapone Capsules Mexiletine Capsules
Flurazepam Capsules Flurbiprofen T ablets Mianserin T ablets Minoeycline T ablets
Fluvoxamine Tablets Folie Aeid Tablets Mitobronitol Tablets M()lJlhine Tablets
Fosfestrol Tablets Furosemide Tablets Moxisylyte T ablets Nabumetone T ablets
Gemfibrozil Capsules Gemfibrozil Tablets Naftidrofuryl Capsules Nalidixic Aeid Tablets
Glibenclamide Tablets Gliclazide Tablets Naproxen Tablets Neomyein Tablets
Glipizide Tablets Gliquidone T ablets Neostigmine Tablets Niclosamide Tablets
Glyeeryl Trinitrate Tablets Griseofulvin T ablets Nicotinamide Tablets Nieotinie Acid T ablets
Guanethidine Tablets Halibut-liver Oil Capsules Nicotinyl Alcohol Tablets Nifedipine Capsules
Haloperidol Capsules Haloperidol Tablets Nimodipine T ablets Nitrazepam Tablets
V -A628 Supplementary Chapter 1 E 2014
Nitrofurantoin Tablets Norethisterone Tablets Sotalol Tablets Spironolactone Tablets

Norfloxacin Tablets Norgestrel Tablets Stanozolol Tablets Sulfasalazine Tablets
Nortriptyline Capsules Nortriptyline Tablets Sulfinpyrazone Tablets Sulindac Tablets
Nystatin Tablets Orciprenaline Tablets Sulpiride Tablets Sumatriptan Tablets
Orphenadrine Oxazepam Tablets Tamoxifen Tablets Temazepam Tablets
Hydrochloride Tablets Tenoxicam Tablets Terbutaline Tablets
Oxprenolol Tablets Oxybutynin Tablets Terfenadine Tablets Tetracycline Capsules
Oxymetholone Tablets Oxytetracycline Capsules Tetracycline Tablets Thiamine Tablets
Oxytetracycline Tablets Paracetamol Tablets Tiabendazole Tablets Timolol Tablets
Dispersible Paracetamol Soluble Paracetamol Tablets Tioguanine Tablets Alpha Tocopheryl Succinate
Tablets Tablets
Paroxetine Tablets Penicillamine Tablets Tolazamide Tablets Tolbutamide Tablets
Pentazocine Capsules Pentazocine Tablets Tramadol Capsules Tranexamic Acid Tablets
Pentobarbital Tablets Perphenazine Tablets Tranylcypromine Tablets Triamcinolone Tablets
Pethidine Tablets Phenelzine Tablets Triamterene Capsules Triflu~erazine Tablets
Phenindione Tablets Phenobarbital Tablets Trihexyphenidyl Tablets Trimethoprim Tablets
Phenobarbital Sodium Phenoxybenzamine Trimipramine Tablets Triprolidine Tablets
Tablets Capsules
Ursodeoxycholic Acid Ursodeoxycholic Acid
Phenoxymethylpenicillin Phenytoin Capsules Capsules Tablets
Tablets
Verapamil Tablets Vigabatrin Tablets
Phenytoin Tablets Phytomenadione Tablets
Warfarin Tablets Zuclopenthixol Tablets
Pimozide Tablets Pindolol Tablets
Acepromazine Tablets Amoxicillin Tablets
Piperazine Phosphate Piroxicam Capsules [BP (Vet)] [BP (Vet)]
Tablets
Ampicillin Tablets Chlortetracycline Tablets
Pizotifen Tablets Poldine Tablets [BP (Vet)] [BP (Vet)]
Polythiazide Tablets Effervescent Potassium Cobalt Depot-tablets Co-trimazine Tablets
Chloride Tablets [BP (Vet)] [BP (Vet)]
Potassium lodate Tablets Pravastatin Tablets Etamiphylline Tablets Lincomycin Tablets
Prazosin Tablets Prednisolone Tablets [BP (Vet)] [BP (Vet)]
Primidone Tablets Probenecid Tablets Methyltestosterone Tablets Phenylbutazone Tablets
[BP (Vet)] [BP (Vet)]
Procainamide Tablets Prochlorperazine Tablets
Piperazine Adipate Tablets Piperazine Citrate Tablets
Prochlorperazine Buccal Procyclidine Tablets
[BP (Vet)] [BP (Vet)]
Tablets
Sulfadimidine Tablets Sulfadoxine and
Proguanil Tablets Promazine Tablets
[BP (Vet)] Trimethoprim Tableis
Promethllzine Hydrochloride Promethazine Teoclate [BP (Vet)]
Tablets Tablets
Tylosin Tablets rBP (Vet21
Propantheline Tablets Propranolol Tablets
Pro1JYlthiouracil Tablets Protriptyline Tablets 3.5 Standardised conditions and limits are considered
appropriate for a pharmacopoeial test that is intended to
Pseudoephedrine Tablets Pyrazinamide Tablets apply to monographs covering products from different
Pyridostigmine Tablets Pyridoxine Tablets manufacturers. It might be argued that non-standardised
conditions and limits would be more discriminatory but
Pyrimethamine Tablets Quinidine Sulfate Tablets
'tailor-made' test conditions and limits may introduce
Quinine Bisulfate Tablets Quinine Sulfate Tablets product bias and may discriminate unnecessarily
Ramipril Capsules Ramipril Tablets between products that are equally acceptable from a
clinical view-point. Similarly with sufficient manipulation
Ranitidine Tablets Rifampicin Capsules of the test conditions, dissolution of almost any product
Ritodrine Tablets Salbutamol Tablets can be achieved. Ideally the test should reflect clinically
significant differences in bioavailability arising from
Selegiline Tablets Senna Tablets differences in dissolution in such a way that clinically
Compound Sodium Sodium Chloride Tablets acceptable formulations will pass whereas clinically
Bicarbonate Tablets unacceptable formulations will fail.
Sodium Fluoride Tablets Sodium Valproate Tablets 3.6 Another issue that has been considered in relation to test
conditions and criteria is that of multiple-point
dissolution protiles as opposed to single-point dissolution individually in consultation with the manufacturers.
tests. It has been concluded that for conventional-release It should be appreciated, however, that the retrospective
preparations such an extension of testing is not general1y addition of dissolution tests is not without its difficulties.
necessary or appropriate for pharmacopoeial purposes. The problems are most acute for those well-established
preparations that are manufactured by a wide range of
4. Function
companies, each with its own dissolution specification.
4.1 The ultimate objective of dissolution testing may be
A pragmatic approach is being taken to developing
described as ensuring adequate and reproducible
compro mise test procedures in these circumstances.
bioavailability without recourse to routine in-vivo testing.
On sorne occasions, this may be achieved by dissolution 6. Bioavailability and Bioequivalence
testing of a particular product for which in vitra/in vivo 6.1 Compliance with the standard British Pharmacopoeia
correlation has been demonstrated. requirement for dissolution provides an assurance that
4.2 A more common objective of dissolution testing is to most of the active ingredient will be dissolved in a
obtain information about the drug release characteristics reasonable amount of time when the preparation is
of a particular formulation or batch of product under subjected to mild agitation.
standardised in vitra testing conditions. 6.2 It should be noted that compliance with the
4.3 Dissolution testing may also be carried out during pharmacopoeial dissolution test do es not by itself
product development studies and is a useful tool in guarantee bioavailability and is not necessarily an
optimising formulation and manufacturing parameters. adequate basis for judging bioequivalence between
It is usually required by tlie Competent Authority as part preparations. Preparations having similar dissolution
of a marketin~ authorisation application. characteristics may not be bioequivalent and vice versa.
4.4 Once a product is licensed, dissolution testing may be 7. Application of harmonised dissolution limits ("Q"
required routinely as part of quality control to values)
demonstrate consistency of manufacture before the 7.1 Since the harmonisation of the dissolution test through
re1ease of each batch of the finished product or, when the Pharmacopoeial Discussion Group and the adoption
necessary, to provide evidence to support changes in of harmonised dissolution criteria ("Q values") between
manufacture such as minor changes in formulation or the European, United States and Japanese
process, changes in site or changes in immediate Pharmacopoeias, the BP Commission has received many
packaging materials. queries regarding the application of limits to dissolution
5. Application tests .
5.1 The British Pharmacopoeia Commission has not 7.2 As an aid to analysts several worked examples are
adopted a policy of universal application of the provided as an illustrative guide below.
dissolution test and, therefore, a dissolution requirement 7.3 An immediate-release tablet formulation has a limit of
will not be included autonÍ.atically in every capsule and Q = 75 %. Six units are tested.
tablet monograph. The International Conference on Unit Result (% of stated amount)
Harmonisation (ICH) guideline on Test Procedures and 1 83
Acceptance Criteria for New Drug Substances and New 2 87
Drug Products (rCH Q6A) contains guidance on the 3 80
application of dissolution testing to drug products and 4 88
this will be used as a basis for deciding whether to 5 90
include a dissolution test in an individual monograph. 6 82
5.2 As a general guideline, it is expected that all new In order to pass SI, all units must be greater than or
monographs for conventional-release capsules and tablets equal to (Q + 5)% (i.e. 80%). Hence, the aboye batch
will contain a dissolution requirement except (i) where passes at SI.
the solubility of the active ingredient at 37 ± OS is
7.4 An immediate-release tablet formulation has a limit of
high throughout the physiological pH range
(dose/solubility volume <250 mL at pH 1.2 to 6.8); (ii) Q = 75%. Six units are tested.
where the dissolution of the dosage form is greater than Unit Result (% of stated amount)
80% in 15 minutes at pH 1.2, 4.0 and 6.8; (üi) where a 1 77
relationship has been determined between disintegration 2 87
and dissolution, or when disintegration has been 3 70
observed to be more discrirninatory than dissolution. 4 88
In circumstances where conditions (i) to (iii) have been 5 90
satisfied, the dissolution test may be replaced by the 6 82
disintegration test lar tablets and capsules, Since units 1 and 3 are les s than (Q+ S) % (i.e. 80%),
Appendix XII A1. the batch fails at S 1. A further six units must then be
5.3 If challenged, a product would be expected to comply tested.
with the test for dissolution specified in the individual
monograph, or if none is specified, with the Test
conditions and acceptance criteria outlined in section 3.
5.4 Dissolution tests have been added to a considerable
number of monographs in the British Pharmacopoeia.
While the objective is to include a 'standard'
pharmacopoeial test wherever appropriate, the
circumstances for each preparation are considered
V-A630 Supplementary Chapter 1 F 2014
Unit Result (% of stated amount) 7.6 An irnmediate-release capsule formulation has a limit of
7 78 Q = 75%. Six units are tested.
8 85 Unit Result (% of stated amount)
9 79 1 83
10 84 2 82
11 87 3 80
12 85 4 79
5 49
In order to pass S2, the mean of twelve units must be
6 77
greater than or equal to Q and no unit can be less than
(Q - 15) % (i.e. 60%). The mean ofthe twelve units is Since unit 5 is less than (Q - 25)% (i.e. 50%), the batch
83% so this passes at S2 and, units 1 and 3, whilst would fail at S3. Testing further units will not enable
failing at SI, are not less than (Q - 15) %, so this batch this batch to pass the test.
passes at S2. 7.7 An immediate-release capsule formulation has a limit of
7.4 An immediate-release tablet formulation has a limit of Q = 75%. 24 units have been tested.
Q = 75%. Six units are tested. Unit Result (% of stated amount)
Unit Result (% of stated amount) 1 76
1 77 2 82
2 87 3 80
3 70 4 79
4 88 5 70
5 76 6 77
6 79 7 65
8 69
Since units 1, 3, 5 and 6 are less than (Q+ 5) %
(i.e. 80%), the batch fails at SI. A further six units must 9 77
10 73
then be tested
11 68
7 73 13 79
8 85 14 65
9 79 70
15
10 84 16 72
11 87 17 67
12 58 18 83
Because unit 12 is less than (Q - 15) % (i.e. 60%) the 19 63
batch fails at S2. A further twelve units must then be 20 68
tested. 21 74
13 79 23 75
14 83 24 70
15 67 All the units are greater than (Q - 15)% (i.e. 60%),
16 84 however; because the mean (73%) is less than Q, this
17 80 batch fails at S3.
18 82
19 78
20 85
21 84 F. Declaration of Content
22 81
This section describes the way in which the content 01 active
23 83 substance in a preparation is declared and the method adopted to
24 88 express the content 01 the medicinal substances themselves.
In order to pass S3, the mean of 24 units must be A proper understanding 01 such statements is essential lO their
greater than or equal to Q , not more than two units can correct interpretation.
be les s than (Q - 15)% (i.e. 60%) and no unit can be
less than (Q - 25)% (i.e. 50%). The mean of the 24 Medicinal substances
units is 80% so this passes at S3. Only unit 12 is less 1. The purpose of the assay in monographs for medicinal
than (Q - 15)% and none are less than (Q - 25)% so substances, taken in conjunction with the tests for impurities,
this batch passes at S3. is to determine the purity of the medicinal substance and the
limits are therefore usually stated in terms of the molecular
entity (salt, ester, etc.) and calculated with reference to the
anhydrous or dried substance as appropriate (depending on
whether the monograph inc1udes a test for water or for loss
on drying).
2. One advantage of this form of expression in 'parent'
monographs is that it gives an indication 'at a glance' of the
purity of the substance. For example (albeit an extreme
example) the purity of Amitriptyline Embonate is stated as
2014 Supplementary Chapter 1 H V-A631
not less than 98.5% of (C2oH23N)2,C23HI 606 calculated 2. This distinction, which is consistent with the approach
with reference to the anhydrous substance rather than as not adopted in the European Pharmacopoeia, is made in
less than 57.9 % of C 2oH 23 N calculated with reference to the recognition of the complexity of the statutory and advisory
anhydrous substance. framework within which the labelling of medicines is
3. The mode of expression chosen for the 'parent' determined. It is hoped that by thus restricting mandatory
monograph in no way circumscribes that which may be used pharmacopoeial labelling statements ro those that are
in the monograph for a preparation. There is no reason why essential for pharmacopoeial purposes, the potential for
the two should be the same and there is frequently good conflict between pharmacopoeial and other statutory
reason why they should be different. In the example of provisions will be minimised.
Amitriptyline Embonate, the assay limits for the preparation 3. Within the context of any particular monograph, it should
Amitriptyline Oral Suspension are stated in terms of be apparent which of the labelling statements are necessary
amitriptyline, C 2o H 23 N in the BP 2000. to demonstrate compliance with the monograph and are thus
mandatory. As guidance, a labelling statement is considered
Forlnulated preparations essential for a medicinal substance
4. The purpose of the assay in monographs for formulated (i) where a test or assay requirement is expressed in relation
preparations is to determine whether the content of the active to a declared value, the 'labelled claim' (for example, the
ingredient is within acceptable limits of the labelled claim apparent viscosity of Carmellose Sodium or the potency
and the limits are therefore of necessity stated in terms of the of Calcitonin (Salmon);
moiety declared on the label as established by the (ii) where different test requirements or limits apply ro
manufacturero materials derived from different sources (for example,
5. Every effort is made in the British Pharmacopoeia to the requirements for matter insoluble in 5M arnmonia
achieve internal consistency within monographs, that is, ro depend on the botanical source of Podophyllum Resin),
use the same terms for content statement, assay and labe!' or intended for different purposes (for example, the
The British Pharmacopoeia Cornmission, however, has no sterility of Benzylpenicillin Sodium when intended for
means of achieving external (inter-monograph) consistency use in the manufacture of a parenteral dosage form
since, unless it perceives there to be a potentially serious risk, without a further sterilisation process);
it would not seek to obtain a change in a manufacturer's (iii) in other special circumstances.
established practice. Problems arise when a manufacturer is
4. Likewise a labelling statement is considered essential for a
not consistent or does not state clearly to what the strength
formulated preparation
refers and, in particular, when different manufacturers of the
same preparation express the content in different terms. (i) where a test requirement is expressed in relation ro a
declared value, the 'labelled claim';
6. Ideally in many cases where several salts or hydrated forms
of the same drug substánce are available, the label and dose (ii) where the content of active ingredient is required to be
(and therefore all monograph stateménts) should be in terms expressed in terms other than the weight of the official
of the anhydrous free base or acid, that is, the active moiety, medicinal substance used in making the formulation (for
in order to facilitate comparison and equivalent dosage. example, Primaquine Tablets contain Primaquine
Phosphate but the content is expressed in terms of the
7. Implementation of such a policy would clearly require that
equivalent amount of primaquine base);
each case should be judged on its merits since there would
be instances when, for example, a different salt is considered (iii) where different test requirements or limits apply ro the
as a different active moiety or where it would be misleading formulated preparation manufactured from different bulk
ro suggest that two different forms are therapeutically drug substances (for example, Heparin Injection
equivalent. Nevertheless it is strongly recommended that as a manufactured from Heparin Calcium or Heparin
general rule lor new drug substances, doses and strengths of Sodium) or intended for different purposes (for example,
preparations should be expressed in terms of the active Isosorbide Dinitrate Tablets intended to be chewed
moiety. before swallowing or allowed to dissolve in the mouth);
8. Meanwhile, for established material s the Pharmacopoeia (iv) in other special circumstances.
will continue to reflect current practice. In this respect it 5. Advisory labelling statements for formulated preparations
should be noted that the labelling requirements of the may be included either in the general monograph or in the
Pharmacopoeia are not comprehensive. Thus a monograph monograph for the individual preparation, as appropriate.
requirement to state the content of active ingredient in terms Such statements commonly relate to features such as the
of the entire drug substance molecule does not pteclude an expiry date, the storage conditions, the name and amount of
additional indication of the content expressed in terms of the any excipients and the directions for making the final
active moiety where such an indication is considered preparations. Additional advisory labelling statements may
desirable. relate to the directions for using the preparation or the
precautions relating to the handling and use of the
preparation.
G. Labelling
This section provides guidance on the status and interpretation 01 H. Biological Assays and Tests
phalmacopoeiallabelling sections. This section provides inlormation and guidance conceming the
l. The General Notice on Labelling distinguishes between biological assays and tests 01 the Pharmacopoeia and the standard
the mandatory status of those Pharmacopoeiallabelling preparations required lor them.
statements that are necessary to demonstrate compliance with l. Biological, including biochemical or immunochemical,
the monograph and the advisory status of other labelling methods are described for the determination of potency or
statements included in the Pharmacopoeia. other specific properties of certain substances and
V-A632 Supplementary Chapter 1 H 2014
preparations where these properties cannot be adequately may be necessary ro obtain the approval of the appropriate
detennined by chemical or physical means. The principie authority for the use of working standards. The biological
applied wherever possible throughout these methods is that properties of the samples selected as working standards
of comparison with a standard preparation so as to detennine should confonn as closely as possible to those of the primary
how much of a sample being examined produces the same standard and an assurance that these conditions have been
effect as a given quantity, the Unit, defined by the standard fulfilled is usually obtained by comparing the behaviour of
preparation. It is an essential condition of such methods that the two samples under varying conditions of comparative
the tests on the standard preparation and on the sample, the testing. In such tests a detailed study of the dose-response
potency or other property of which is being detennined, shall curves may indicate whether the sample selected to serve as a
be carried out at the same time and, in all other respects, working standard is a suitable preparation. For the assay of
under strictly comparable conditions. certain biological materials in the Phannacopoeia, for
2. Standard Preparations, as defined in the biological assays example oxytocin, European Phannacopoeia Biological
and tests of the Phannacopoeia, are of two kinds: primary Reference Preparations have been established and are
standards which are established, held and distributed by the recommended for use as the working standards. Such
appropriate intemational oronational organisation and preparations may be obtained from the European
secondary (working) standards which are preparations the Phannacopoeia Commission (address as aboye).
potencies of which have been detennined by an adequate 5. Wherever possible the primary standard is the
number of comparative tests in relation to the relevant Intemational Standard, and the biological activity is
primary standard. expressed in International Units (IU). In other cases, where
3. A primary standard is a selected representative sample of Units are referred to in the official assays and tests, the Unit
the substance for which it is to serve as a basis of for a particular substance is, for the United Kingdom, the
measurement. It is essential that primary standards shall be specific biological activity contained in such an amount of the
of uniform quality and as stable as possible. These conditions respective primary standard as the appropriate international
are usually ensured by providing the preparations in the dry or national organisation indica tes. The necessary infonnation
sta te, dispensing them in sealed containers free from is provided with the samples of the primary standard.
moisture and oxygen and storing them continuously at a low 6. For enzymes in the Phannacopoeia, where the assay is
temperature and in the absence of light. such that the stoichiometry of the reaction is known, for
For the majority of biological assays of the Phannacopoeia, example, where the substrate is a synthetic ester, the activity
the primary standards are the Intemational Standards and is measured in microkatals or nanokatals. A micro ka tal is
Reference Preparations, established by the World Health defined as the enzyme activity that under defined conditions,
Organization. produces one micromole of the reaction product per second
Laboratories in the United Kingdom may obtain these for or altematively consumes one micro mole of the reaction
the purposes of the biological assays described in the substrate per second. For other enzymes in the
Phannacopoeia from the National Institute for Biological Phannacopoeia, where the reaction involved in the assay is
Standards and Control, Blanche Lane, South Mimms, more complex, for example, where the substrate is a naturally
Potters Bar, Hemordshire EN6 3QG, England. occurring macromolecule such as a protein, the activity is
measured in Units as previously defined.
In October 2005 the European Directorate for the Quality of
Medicines & HealthCare (EDQM) was designated as the 7. The methods of biological assay described in the
Intemational Collaborative Centre for the establishment and Phannacopoeia have been found satisfactory but will not
distribution of WHO International Standards for Antibiotics necessarily be the best methods for use in all circumstances.
(ISA) , replacing the National Institute for Biological In most instances they may be replaced by other methods if
Standards and Control (NIBSC) in this role. These it can be shown that such methods are at least equally
standards may be obtained from the Sales Section, EDQM - accurate and precise and .provide a measurement of the same
Council of Europe, 7 allée Kastner, CS 30026, 67081 active principies.
Strasbourg, France. Further infonnation can be found on the 8. Any estima te of potency derived from a biological assay is
EDQM website (www.edqm.eu). subject ro random error due to the inherent variability of
For the assay of certain enzymes in the Phannacopoeia the biological responses and calculations 6f error should be
Standard Preparations, as defined in the appropriate method, made, if possible, from the results of each assay and recorded
are the primary standards established by the International with the potency estimate even when the official method of
Commission on Phannaceutical Enzymes of the Intemational assay is used. Guidance on the design of assays, the statistical
Phannaceutical Federation (FIP). These standards may be analysis of results and the calculation of potency is provided
obtained from the Centre for Standards, Wolterslaan 12, in general text 5.3 of the European Phannacopoeia
B- 9000, Ghent, Belgium or, where these standards have (Supplementary Chapter IV G) . The methods described
been established in co-operation with, and adopted as official therein take account of the inherent random error but
preparations by, the European Pharmacopoeia Commission, assume that systematic errors, for example, errors in weighing
they may be obtained from the EDQM address shown aboye. or dilution, will not represent a major source of variation in
the potency estimates. Alternative assay designs and methods
4. As a measure of economy in the use of the primary
of calculation may be used provided that they are not less
standards it is recommended that working standards should reliable.
be prepared and used in those biological methods of the
Phannacopoeia where the definition of the Standard 9. Where an irnmunoassay, that is an assay procedure based
Preparation is so worded as to pennit this. However, in sorne on the reversible and non-covalent binding of an antigen by
instances the complexity or lack of precision of the method antibody, is described in the Phannacopoeia for detecting or
or difficulties associated with the preparation of a secondary quantifying either an antigen or an antibody, the
standard render such a practice inadvisable and in any considerations described in Appendix XIV B apply in
country in which a particular assay is controlled by law it addition to the general points referred to aboye.
2014 Supplementary Chapter 1 J V-A633
8. As noted in paragraph 2 aboye, the pharmacopoeial test is

J. Efficacy of Antimicrobial Preservation intended to provide a framework within which to develop a
This section provides background informatio71 071 the purpose and test suitable for the particular product being examined.
scope of the test for efficacy of antimicrobial preservation. Detailed descriptions of all aspects of testing are therefore
The Annex to this section provides guidance on some practical inappropriate within the pharmacopoeial test itself.
aspects of testing. The following Annex draws attention to sorne key features of
l. The test for efficacy of antimicrobial preservation, the test and offers additional guidance on certain practical
ineluded in Appendix XVI C, has been accorded non- aspects of testing.
mandatory status in the Pharmacopoeia. This status is
reftected in the test' s inelusion as a general text in section 5 Annex
of the European Pharmacopoeia and in the form of reference 9. Several features have been identified as sources of
to the test within Production sections of relevant general procedural variation in the test that may contribute to
monographs of the European Pharmacopoeia .. Non- variation in the outcome of the test. A elearly written and
mandatory status provides a degree of ftexibility consistent sufficiently detailed test protocol is therefore essentia!. Sorne
with the intended purpose of the pharmacopoeial test. of these features are discussed in this Annex.
2. The pharmacopoeial test is intended to serve as a model 10. Test organisms; culture maintenance Potential
offering a manufacturer guidance conceming this aspect of sources of variation to which attention should be given
quality and a foundation on which he can build to meet his inelude the source, age and storage of the microbial culture
own particular needs. The testing procedure is intended to and the number of passages between the freeze-dried strain
serve as a means whereby, during product development, a collection culture (repository culture) and the suspension of
manufacturer can assess the efficacy of any antimicrobial organisms used to inoculate the product (the test
preservative ineluded in the producto Within the context of suspension). It is recommended that the repository culture is
the British Pharmacopoeia, Appendix XVI C serves to define cultured and aliquots freeze-dried or stored under liquid
what is meant by the term 'imitable antimicrobial nitrogen. It is also recommended that the sub-culture used as
preservative' in accordance with the relevant Notice in Part 2 the test suspension should be no more than 5 passages from
of the General Notices. the repository culture.
3. If during developmént a fully quantitative, comparative 11 . Product inoculation Potential sources of variation to
evaluation of diff~rent preservative systems would be useful, which attention should be given inelude the culture media
the procedure described in the Appendix could be extended. and method used to prepare the inoculum, the
For example, the incorporation of additional sample times inoculum:product ratio and the homogeneity of the
would allow an estimation of microbial death rates. inoculum-product mixture. Appendix XVI C sta tes that the
4. The test is intended for application to those products that test suspension shall be used immediately. It is recommended
may support the growth or viability of microbial that this is interpreted to mean that, once prepared, the test
contaminants. It is based on the premise that preservatives suspension should not be sto red but may be used over a
are used primarily to protect products from in-use microbial period of 8 hours provided that it is kept at a temperature of
contamination but also recognises their role in reducing the 2° to 8°.
bioburden of 'non-sterile' products. In-use contamination is 12. Test container It is emphasised that, as indicated in the
of relevance to multidose products, both sterile and Appendix, wherever possible, the product should be
non-sterile. Bioburden is of relevance to non-sterile products, inoculated andoincubated in its original container (market
both multidose and single dose. pack). Where, however, it is necessary to transfer the product
S. The present European Pharmacopoeia text provides to sorne other container, for example, in order to achieve
criteria for parenteral, ophthalmic, topical and oral homogeneous distribution of the inoculum, careful
preparations . The criteria for ear preparations are equivalent consideration needs ,to be given to the choice of test
to those specified for topical preparations thus maintaining container. Features of such a container that will inftuence its
parity between these two types of producto suitability as a test container inelude the material from which
6. An unusual feature of the European Pharmacopoeia text is it is made, its shape and size (volume) and the type of
the inelusion of two sets of criteria (A and B) for parenteral elosure . The material from which the container is made
and ophthalmic preparations and for topical preparations. should not affect the product, for example, by leaching or by
The A criteria express the recommended efficacy to be sorption of ingredients. Particular attention should be paid ro
achieved, that is, they represent generally applicable 'target' possible changes in product pH since these can markedly
criteria. affect preservative activity. A container with smooth surfaces
and a wide neck that will allow ease of access and mixing is
7. It is recognised that for certain products, for example,
recommended for creams and other viscous preparations.
some antacids and certain biological products, these target
criteria are unlikely to be achieved except at the expense of 13. Test procedure Test conditions such as storage
some other property of equal or greater importance. temperatures and sample times are given in
The altemative criteria to be met in these circumstances are a Appendix XVI C. Working at a temperature within the stated
matter for agreement between the manufacturer and the range of 20° to 25°, it is advisable, wherever possible, to
competent authority and should take account of any special minimise the temperature difference from test to test.
considerations relevant to the specific producto The B criteria 14. Counting survivors The Appendix specifies that the
for parenteral and ophthalmic products and for topical count obtained for the inoculum is used as the baseline for
products were adopted by the European Pharmacopoeia calculating the reduction in viable micro-organisms .
Commission in deference to those member sta tes that wanted 15. Preservative inactivation When residual antimicrobial
published guidance on the minimum values below which any activity is removed either by the use of an inactivating agent
altemative criteria should not fal!. or by dilution, it is necessary to confirm the ability of the
system to support the growth of the test organisms by the use
V -A634 Supplementary Chapter 1 K 2014
of appropriate controls. These controls should simula te test their single specific configuration and the phenylmalonyl side-
conditions and allow validation of the counts. In designing chain chiral atom marked with an asterisk.
such controls an appropriate target recovery efficiency, for
example 70% recovery, should be seto Tests
16. Interpretation of results Where a test criterion is given 7. In future, when a monograph describes an enantiomer, it
as 'no increase' this is intended to be interpreted as no will inelude both a test for specific optical rotation under
increase aboye the counts obtained at the previous specified Identification and a test, using m ethods such as chiral
sample time. It is expected that, having achieved the required chromatography, to control enantiomeric purity.
reduction in counts at the shorter time interval specified, the 8. When both the racemic mixture and the enantiomer are
preservative will maintain the microbial population at or available, the monograph for the racemic mixture wilI, where
below this lower leve!. appropriate, specify a test for angle of rotation together with
a cross reference under Identification. The test for angle of
rotation will normally specify low, symmetrical limits about
K. Stereochemistry zero where this has been shown to limit the presence of
optically active impurities and demonstrate approximately
This seccion describes lheway in which lhe stereochemislry of a equal proportions of the enantiomers.
subsrance is indica red in lhe chemical definirions and graphic
fonnulae of lhe Brüish Phannacopoeia and lhe way ir may be 9. When only the racemic mixture is available, the
idenrified and/or conrrolled within the tests in a monograph. monograph for the racemic mixture may, where appropriate,
specify a test for angle of rotation.
l. Many medicinal substances that contain one or more
chiral centres and that are already on the market have been
made available for pharmaceutical use as racemic mixtures
with little known about the biological activities of the
separate isomers. This has been refiected in the monograph
L. Microbiological Assay of Antibiotics
in the Pharmacopoeia and a test to show that the substance This section provides guidance on inrerprelalion of statements in
is the racemic mixture has not usually been ineluded unless it the Pharmacopoeia concerning content limits for those antibiotics
was known that at least one of the separate enantiomers was and their preparations for which the monograph specifies a
. also available commercially. Nevertheless, with increasing microbiological assay .
concem by r~gulatory authorities for substances to be made l . The statements in the Pharmacopoeia concerning the
available as single isomers, tests for enantiomeric composition potency of antibiotics are framed to provide a control analyst
wilI become more common (see section on tests below). with the means whereby he can ascertain whether or not the
material or preparation in question is satisfactory, that is,
Chemical definition they provide criteria appropriate to a 'check assay'.
(monographs other than those of lhe European Phannacopoeia) 2. In order to be confident that, when assayed by a control
2. In the case of substances containing a single chiral centre, analyst, a material or preparation would meet the
the descriptor '(RS)-' is ineluded at the appropriate position pharmacopoeial criteria a manufacturer would himself need
in the chemical definition of the substance to indicate a to work to criteria appropriate to a 'release assay'.
racemic mixture. 3. The need for different criteria for check and release assay
3. For substances containing muItiple chiral centres and purposes arises from the inherent variability of biological
comprising a mixture of all possible stereoisomers the term systems which precIudes assigning an exact value to the true
'all-rac-' has been used, for example Isoaminile. In those few potency. Ir is possible only to give a range of values within
substances existing as diastereoisomeric mixtures, that is which the true potency can' be expected to lie with a defined
where in one or more centres the stereochemistry is explicit degree of confidence (95%; P = 0.95 for pharmacopoeial
but in other centres it is not, each centre is d.efined either as purposes). In such circumstances the fiducial limits of error
the specific (R)- or (S)- configuration, or as racemic computed for the assay in question define the lower and
(RS)-, respectively. upper limits within which the true potency of the sample lies,
with a probability of 95%.
Graphic fonnulae
4. The required minimum precision for an acceptable assay
4. When a medicinal substance is a racemate, an indication is of any particular antibiotic or preparation is defined in the
given by means of the graphic formula . appropriate monograph in the paragraph entitled Assay. This
5. Because in graphic formula e there is no generally accepted degree of precision is the minimum acceptable for
convention for depicting a racemate, each racemic substance determining that the final product complies with the official
with one chiral centre is shown in the (R)- form with the requirements. It may be inadequate for a decision about the
appended text 'and enantiomer' for example, Carteolol potency that should be stated on the label or used as the
Hydrochloride. For the all-rac- mixtures, such as Docusate basis for caIculating the quantity of an antibiotic to be
Sodium and Alpha Tocopheryl Acetate, non-stereospecific incorporated in a preparation. In such circumstances, assays
graphic formula e are drawn and the legend 'mixture of n of greater precision may be desirable with, for instance,
stereoisomers' added beneath (where n is the number of fiducial limits oferror ofthe order of98 to 102%. With this
possible stereoisomers); the stereogenic carbon atoms degree of precision, the lower fiducial limit lies elose to the
concerned are identified by means of asterisks. estimated potency. By using this limit, instead of the
6. In diastereoisomeric mixtures, the unique configuration estimated potency, to assign a potency to the antibiotic either
centres are drawn as such, while each racemic centre (with for labelling or for caIculating the quantity to be incIuded in
equal amounts of the (R) and (S) configuration) are a preparation, there is less likelihood of the final preparation
indictated by an asterisk and the legend 'racemic at C*' subsequently failing to comply with the official requirements
appended. For example, Carbenicillin Sodium is drawn in for potency. Greater precision can be achieved by statistically
this way; the chiral atoms in the penicillanic acid ring each in combining the results of two or more independent assays.
2014 Supplementary Chapter 1 L V-A635
Bulk antibiotic than 90% or if the lower fiducial limit of error was greater
5. In a monograph for a bulk antibiotic a minimum potency than 115 % of the amount expected in accordance with the
in IV is given under the heading Definition and this is to be strength stated on the labe!' A manufacturer, on the other
interpreted in accordance with the second paragraph of the hand, would consider suitable for release only those batches
General Notice on Biological Assays and Tests. This states of the product for which the lower fiducial limit of error
that 'The material is not of pharmacopoeial quality if the obtained in his assay was greater than 90% and for which the
upper fiducial limit of error is less than the stated potency. upper fiduciallimit of error was less than 115% of the amount
For such antibiotics the required precision of the assay is expected in accordance with the strength stated on the labe!'
stated in terms of the fiduciallirnits about the estimated Formulated preparation (Fig. SCIL-2)
potency.' Probability = 0.95,
6. Taking Amphotericin, the monograph for which has a Precision = 95 - 105%
defined minimum potency of not les s than 750 IV per mg, as
Let Y be the stated content/strength [labelled clairn] of the
an example of a bulk antibiotic: a control analyst would
preparation [3500 IV per g of ointment, for example, for
judge the material unsatisfactory with respect 10 potency only
Neomycin Eye Ointrnent].
if the upper fiduciallimit of error [UFLE] obtained in his
assay was less than 750 IV per mg, calculated with reference Let R¡, R2 , R 3 , ~, Rs be the estimated content/strength
to the dried substance. A manufacturer, on the other hand, found by the manufacturer in the release assay.
would consider suitable for release only those batches for To ensure (with 95% confidence) that the true
which the lower fiducial lirnit of error [LFLE] obtained in his content/strength is not less than the specified minimum [ie
assay was greater than 750 IV per mg, calculated with that R not < 90% Y), the manufacturer releases only batches
reference to the dried substance. such as Rl and R 2 for which the lower fiduciallimit of error
of the estimated potency is 2: 90% Yi he rejects batches such
Bulk antibiotic (Fig. SClL-l)
as R3 and ~ for which the lower fiducial limit of error is
Probability = 0.95, ' < 90% Y. To ensure (with 95% confidence) that the true
Precision = 95- 105% content/strength is not greater than the specified maximum
Let X be the monograph value for the minirnum potency of [íe that R not > 115% Y), the manufacturer also rejects
the bulk antibiotic [750 IV per mg, for example, for batches such as Rs for which the upper fiduciallimit of error
Amphotericin] . is > 115% Y. In order to avoid possible problems of
Let R¡, R2 , R3 be the estimated potency found by the compliance with monograph assay limits at the end of
manufacturer in the release assay. shelf-life for those antibiotics where stability is an issue,
manufacturers are advised to exercise caution in releasing
To ensure (with 95% confidence) that the true potency of his
batches such as R2 for which the lower fiducial limit of error
bulk substance is not less than the desired minimum [ie
is les s than the value stated on the labe!' The advice given in
that R not <X], the manufacturer releases only a batch such
paragraph 4 may be of assistance in these circumstances.
as R¡ for which the lower fiducial lirnit of error of the
estimated potency is 2:Xi he rejects batches such as R2 Let Cl' C2 , C 3, C4 , CS, C6 be the estimated content/strength
and R3 for which the lower fiducial limit of error is <X. found by the control analyst in the check assay.
Let Cl' C2, C3 , C4 be the estimated potency found by the Only when the lower fiducial lirnit of error of the estirnated
control analyst in the check assay. content/strength is > 115% Y or when the upper fiducial
limit of error of the estimated content/strength is < 90% Y
Only when the upper fiduciallirnit of error of the estirnated
can the control analyst conclude (with 95 % confidence) that
potency is < X can the control analyst conclude (with 95%
the true content/strength is outside the specified rangei
confidence) that the true potency of the sample being
he would fail batches with estirnated content/strength C 4 and
examirted is less than the desired minimum value. He would
fail a batch with estimated potency C 4 . C6 ·
It is important to recognise that, for the reasons explained in
Fonnulated preparation paragraph 3, a batch giving the estimate ~ in a release assay
may give the estimate C4 in a check assay without any change
7. In a monograph for a formulated preparation the lower
in the true potency.
and upper lirnits of content are given under the heading
Assay and are given as the values (statedin terms of Definitions
percentage of labelled claim) within which the fiduciallimits
of error of the estimated potency obtained in the Assay must Assay A single test carried out for the purpose of
lie. estimating the potency of a material/preparation or the
pooled results of two or more such tests.
S. For a formulated preparation the results of the Assay and
the content, or strength, stated on the label are given in True potency The actual potency of the
terms of IV unless specific instruction 10 calculate a 'weight material/preparation at the time of assay. In practice this
equivalent' is given as, for example, in the monograph for value can never be exactly evaluated.
Strep10mycin Injection. Such a 'weight equivalent' cannot be Stated or labelled potency This is often a nominal value
given for certain antibiotics, in particular those like Neomycin assigned to a formulated preparation from knowledge of the
Sulfate which consist of a variable mixture of components. potency of the bulk materia!. In the case of bulk materials, it
9. Taking Neomycin Eye Ointrnent, the monograph for may be calculated from assay data.
which requires the upper fiduciallirnit of error 10 be not less Estimated potency The potency calculated from assay
than 90% and the lower fiduciallimit of error 10 be not more data. Ir is misleading to refer to a 'best estimate' [se e under
than 115% of the stated amount, as an example of a fiduciallirnits below] .
formulated preparation: a control analyst would judge a Fiduciallirnits [confidence interval] These are limits 10
product unsatisfactory with respect to content only if the upper the true potency of the material/preparation and are
fiduciallimit of error obtained in the check assay was less calculated on the assumption that there is no bias in the
V-A636 Supplementary Chapter 1 M 2014
[Xl
LFLE UFLE
LFLE elI ULFE
Fig. SC1L-l
90% Y M 115%Y
···
I
LFLE R,
1 UFLE
I···::
: R3
I 1
.
·:
I I
I
I LFLE UFLE :
I
:.
·
I
I I
:: __________C2 I
I
.:
L-____ ~ __ I
:"
i
C3
1 9!
5
_________C6
1___ _
.1.
Fig. SC1L-2
assay system. They may be calculated for any desired level of The use of these two tenns does not imply a fundamental
probability; for phannacopoeial purposes a leve! of 0.95 is difference in the design or execution of an assay but rather
applied. The concept is that, if the assay could be repeated in the interpretation of the results obtained. A check assay
many times, the true potency would lie within the fiducial only enables a control analyst to say unequivocally when a
limits in 95% of assays. Thus, in any particular assay, the material/preparation is unsatisfactory [see illustration abovel.
true potency could be anywhere within the fiducial range
(and occasionally outside it) and the estimated potency is no
better measure of the true potency than any other value in
the range. M. Microbial Contamination
Release and check assays A release assay is an assay This section of Supplementary Chapter 1 provides information on
carried out by those responsible for assigning a potency to a the status and application of the texts on microbial contamination
material or declaring a content or strength for a preparation included in Appendix XVI. These texts, which are taken from the
[manufacturersl. A check assay is an assay carried out by European Pharmacopoeia (Ph Eur), are used both to satisfy
those responsible for checking the potency of a material or mandatory requirements of the Pharmacopoeia and as guidelines
the content or strength of a preparation [control authorities, for the purposes of microbiological monitoring. This chapter
independent analysts and purchasers of bulk materialsl . provides clarification on the application and interpretation of the
test methods and criteria in the different situations.
2014 Supplementary Chapter 1 N V-A637
l. Appendix XVI B and Appendix XVI F comprise several 9. As indicated in the general monograph for Pharmaceutical
sub-sections describing tests for detecting different types of preparations, recommendations on the microbiological
microbial contamination and Appendix XVI D and quality of non-sterile pharmaceutical preparations are
Appendix XVI G provide guidance on the microbial quality provided in Appendix XVI D and Appendix XVI G of the
of pharmaceutical preparations and herbal medicinal BP.
products. The text on Microbiological Quality of Non-sterile
2. The tests for specified micro-organisms, included as Pharmaceutical Preparations, included as Appendix XVI D, is
Appendix XVI Bl, are those included as method 2.6.13 of general text 5.1.4 ofthe Ph Eur. As described in the General
the Ph Eur. These tests describe the methods used to set Notices: 'General chapters become mandatory when referred
mandatory requirements in monographs for certain bulk to in a monograph, unless such reference is made in a way
materials of natural origino For example, the monograph for that indica tes that it is not the intention to make the text
Dried Aluminium Hydroxide specifies the absence of referred to mandatory but rather to cite it for information'.
Escherichia coli and from bile-tolerant gram-negative bacteria Where general text 5.1.4 is referred to in a general
and that for Pancreatin specifies 1 g is free from Escherichia monograph as giving recommendations on microbiological
coli and 10 g is free from Salmonella. These methods are also quality, different acceptance criteria may be justified.
used to SUPPQrt the relevant non-mandatory The general monograph for Oral Liquids, for example, has a
recommendations made within the guidelines on the mandatory Production requirement that 'In the manufacture,
microbiological quality of pharmaceutical preparations and packaging, storage and distribution of liquid preparations for
herbal medicinal products (se e paragraph 9 below). oral use, suitable measures are taken to ensure their
3. The tests for total viable aerobic count, included as microbial quality' the following statement 'recommendations
Appendix XVI B2, are those included as method 2.6.12 of on this aspect are provided in the text on Microbial Quality of
the Ph Eur. These quantitative tests are used in two contexts Phannaceutical Preparations (5. 1.4)' clearly constitutes a non-
within the Ph Eur. In addition to being invoked to set mandatory recommendation.
mandatory limits fo~ a range of bulk materials of natural The text on Microbiological Quality of Herbal Medicinal
origin, the methoas are also used to support the Products for Oral Use, included as Appendix XVI G, is general
non-mandatory guidelines on the quality of pharmaceutical text 5.1.8 of the Ph Eur. This text is described as
preparations and herbal medicinal products (see paragraph 9 'recommended acceptance criteria for microbiological quality
below). of herbal medicinal products'.
4. In Appendix XVI B2 of the British Pharmacopoeia (BP), 10. The texts of Appendix XVI D and Appendix XVI G are
an introductory paragraph sta tes that the tests are designed intended to provide guidance to those concemed with this
primarily to determine whether a substance or preparation important aspect of quality assurance of final dosage forms
complies with an established specification for microbiological and herbal medicinal products for oral use . The advisory
quality. For example, the monographs for Agar and for Dried limits for products in both texts should be used by
Aluminium H ydroxide specify total aerobic microbial counts manufacturers for process validation and may be used as part
of not more than 10 3 micro-organisms per gram and total of a general microbial monitoring programme to identify
combined yeastlmould counts of not more than 10 2 needs for corrective action. The availability of authoritative
micro-organisms per gramo The method indic::ates that, when guidelines provides a useful yardstick for official control
used for such purposes, it is to be carried out in accordance laboratories when conduding surveys of products on the
with the instructions in the general text, including the market. It is emphasised that the inclusion of advisory limits
number of samples to be taken, and the results are to be in the Pharmacopoeia is not intended to imply the need for
interpreted as stated. end-product microbiological testing on a batch-to-batch
5. When the tests are used by a manufacturer for monitoring basis. Such testing would be inconsistent with the principies
raw materials andlor finished products or for process of good manufacturing practice and the analytical burden
validation, the sampling plans for microbiological examination imposed by such testing could not be justified for the
and the method of interpretation of the results are matters for majority of products .
agreement between the manufacturer and the competent
authority. (Se e also Basis of Pharmacopoeial Requirements in
the Introduction to Supplementary Chapter 1.)
N. Particulate Contamination
6. The test for absence of mycoplasmas, included as
This section of Supplementary Chapter 1 provides information 012
Appendix XVI B3, is method 2.6.7 ofthe Ph Eur as applied
the status and application of the texts on particulate contamination
to vaccines for human use. This test is invoked as a
included in Appendix Xl11. Guidance is also offered on limits for
mandatory requirement in the relevant parts of the
visible particles in small-volwne ir¡jections.
Production section of certain monographs for viral vaccines
produced in cell cultures or in eggs, for example, Inactivated l . Appendix XIII comprises two methods for determining
Influenza Vaccine (Surface Antigen). (The test as applied to particulate contamination:- Sub-visible particles and Visible
veterinary vaccines is reproduced in the BP (Veterinary).) particles. Of these two methods, only that for sub-visible
particles is currently invoked as a mandatory requirement
7. The test for mycobacteria, included as Appendix XVI B4, is
within the monographs of the Pharmacopoeia.
method 2.6.2 of the Ph Eur. Reference to this method is
made under the tests for extraneous agents in viral vaccines 2. The test for sub-visible particles included as
(see paragraph 8 below). Appendix XIII A is method text 2.9.19 of the European
Pharmacopoeia. The criteria are invoked by means of the
8. The tests for extraneous agents in viral vaccines, included as
revised Ph Eur general monograph for Parenteral
Appendix XVI B5, are those included as method 2.6.16 of
Preparations . The statements previously included in certain
the Ph Eur. These tests are invoked as mandatory
individual monographs of the British Pharmacopoeia such as
requirements in the relevant parts of the Production section
Sodium Chloride Intravenous Infusion have therefore been
of certain monographs for viral vaccines.
deleted and reliance placed on the general monograph.
V-A638 Supplementary Chapter 1 O 2014
3. The test for visible partic1es included as Methodology for Fine partic1e dos e testing will be inc1uded
Appendix XIII B is method text 2.9 .20 of the European in monographs for inhalation powder and pressurised
Pharmacopoeia. This text describes standardised viewing inhalation in future.
conditions but sets no criteria of acceptance. Contamination Assay methods in inhalation powder and pressurised
by visible particles is governed instead by the requirement of inhalation monographs will be revised to assay a samp1e from
the Ph Eur general monograph for Parenteral Preparations the finished product instead of assaying discharged doses and
that injections and intravenous infusions that are solutions therefore the content of active ingredient delivered by
are required to be 'c1ear and practically free from partic1es' . actuation of the valve sampling method will be discominued
It is recognised that this latter requirement can give rise to in the BP 2015.
problems of interpretation. These problems could, perhaps,
be overcome by providing simple criteria for the test for
visible partic1es suitable for application as a pharmacopoeial
'check-test', that is, for application by an independent
analyst, for example, a hospital quality control pharmacist, as
a means of judging the quality of a particular parenteral
preparation.
4. The need to set' limits 'is based on the premise that an
expectation of total absence of visible partic1es from all
containers from a batch of an injectable preparation is
unreasonable and unrealistic. An attributes-based test using a
small sample together with simple pass/fail criteria would be
consistent with the needs and constraints of a check test.
The criteria chosen would need to be capable of detecting a
batch that was grossly contaminated while representing an
acceptably low chane e of condemning batches of satisfactory
quality through a small sample being unrepresentative.
It would, of course, be unlikely that, if a sample failed such a
test, consequent action would be initiated by the competent
authority on the result of a single test. In such circumstances
the competent authority would investigate the occurrence
further with the manufacturer.
5. The following critena are offered as generally suitable for a
check test for small volume injections that are solutions.
For guidance, it is suggested that in total 20 containers are
examined as described in Appendix XIII B and any partic1es
recorded. It is suggested that the preparation being examined
should be considered to have failed the test if one or more
partic1es are found in more than one container.
6. It is emphasised that these criteria are not intended for use
by a manufactu~er for batch release purposes. It is expected
that a manufacturer would obtain assurance of the quality of
his product with respect to visible particulate matter by
100% inspection or by other appropriate means in
accordance with good pharmaceutical manufacturing practice
(GMP).
O. Inhaled Products
Fonnulated Preparations: Specific Monographs
The tests inc1uded in monographs for inhaled products are
those tests indicated in Supplementary Chapter III C.
Monograph Development: Guidance to Manufacturers.
Methods and limits, where appropriate, are also provided for
tests mandated by the Ph. Eur. general monograph for
Preparations for Inhalation.
The BP requirement for Deposition of the emitted dose test
using Appendix XII C 7 Apparatus A has been discontinued
in the BP 2014 as it has been superseded by appropriate
improved collection apparatus. The BP advocates declaration
of the delivered dos e on product labels in line with the
Ph. Eur. requirements. Where dosing has been established
based on the emitted dos e, both the delivered dose and
emitted dose should be stated on the labe!'
2014 Supplementary Chapter II B V-A639
Pharmacopoeia 1998 in the form 'When [former title) is

Supplementary prescribed or demanded, [current tide) shall be dispensed or
supplied.'. These statements were deleted from the British
Chapter II Pharmacopoeia 2003.
3. For substances for which there was a rINN, it was
necessary to omit any subsidiary tides from the British
Names of Medicinal Substances and Pharmacopoeia 1998. For example, the 'Vitamin C'
Preparations subsidiary titles were omitted from the monographs for
Ascorbic Acid, Ascorbic Acid Injection and Ascorbic Acid
This Supplementary Chapter pro vides information on
Tablets. Statements of the type described aboye
Pharmacopoeial monograph titles, on the construction of
(incorporating the na me used in the former subsidiary title)
tides for formulated preparations and on the structures and
were inc1uded and have been retained in subsequent editions,
nomenc1ature of substances of natural or semi-synthetic
where appropriate, to provide continuity.
origino
4. With the exception of Adrenaline and Noradrenaline, the
Introduction practice of 'dual-Iabelling' (rwo names presented on separate
British Approved Names are devised or selected by the lines) was discontinued in the British Pharmacopoeia 2003.
British Pharmacopoeia Commission and published by the In order to comply with EC Directive 92/27/EEC, the titles
Health Ministers on the recommendation of the Commission of all such affected monographs were replaced by the rINNs.
on Human Medicines. British Approved Names [BANs) are A statement in the form ' The name Uormer tille} was formerly
nonproprietary names established inter alia to provide suitable used in the United Kingdo117' was inc1uded in all affected
tides for monographs for medicinal substances in the monographs in the British Pharmacopoeia 2003 and
Pharmacopoeia. The issue of a British Approved Name, subsequent publications. These statements have been deleted
however, do es not imply that the substance will necessarily from the British Pharmacopoeia 2009.
be inc1uded in the Pharmacopoeia. The guiding principies 5. Adrenaline and Noradrenaline are the terms used in the
that are used in devising or selecting new British Approved tides of monographs in the European Pharmacopoeia and are
Names are stated in the current edition of the British thus the official names in use in the 37 Member States party
Approved Names book. to the Convention on the Elaboration of a European
Where a Recommended Intemational Nonproprietary Name Pharmacopoeia.
[rINN) has been established by the World Health
Organization, this name is now invariably adopted as the
BAN and used as the tide .of the monograph, if any, in the
Pharmacopoeia. In the past there were occasional differences B. Monograph Titles for Formulated
between the rINN and the corresponding BAN. Many, but Preparations
not all, of these differences were minor changes in spelling to
. l. The tides of monographs for formulated preparations
accommodate normal English pronunciation and usage.
combine the appropriate drug substance na me and
Implementation of Directive 92/27 IEEC, however, requires
pharmaceutical formo
the use of rINN in the labelling of medicinal products
throughout the member states of the European Community. 2. The drug substance na me is a British Approved Name
In consequence, the relevant BANs have been modified to [BAN] which in general is also the recommended
accord with the English version of the rINN. These changes Intemational Nonproprietary Name [rINN) . In cases where
were made by means of Supp1ement No. 4 to British the drug substance name is a British Approved Name
Approved Names 1997 and were reflected, where relevant, in (Modified), the modifying term is omitted from the title for
the titles of the monographs in the 1998 edition and the formulated preparation except where there exist rwo or
subsequent editions of the Pharmacopoeia as described in more formulations containing different forms (salts, hydrates
section A of this chapter. etc.) of the drug substance. In such cases the modifying term
is retained in the monograph title. Thus, for example, the
The titles of monographs for medicinal preparations combine
title of the monograph for tablets containing Clonidine
the British Approved Name with an appropriate term for the
Hydrochloride is Clonidine Tablets since tablets containing
dosage form in question as described in section B of this
c10nidine are formulated only with the hydrochloride.
chapter.
The tide of the monograph for tablets containing Isosorbide
Dinitrate, on the other hand, is Isosorbide Dinitrate Tablets,
since tab1ets formulated with the mononitrate are also
A. Changes in Monograph Titles available .
l. Certain changes in monograph titles were made in the 3. The terms for the pharmaceutical form are either (i)
British Pharmacopoeia 1998 in order to bring the titles in the derived from the relevant General Monograph; or (ii) terms
British Pharmacopoeia in line with the names (recommended in established use in the UK.
Intemational Nonproprietary Names [rINNs)) that 4. When the pharmaceutical form is described as the term in
manufacturers are now required to use on product labels and established use in the UK, reference to the pharmaceutical
leaflets in accordance with EC Directive 92/27/EEC. form from the relevant General Monograph will be provided
2. Normal practice within the British Pharmacopoeia when as a Subsidiary Title and incorporated into the Definition
changing the tide of a monograph is to reta in the former tide section of the monograph. For Codeine Linctus, for examp1e,
as a subsidiary title for a period of at least five years. This the tide of the monograph and therefore the nonproprietary
was not possible for these changes since use of the former name of the preparation remains as Codeine Linctus, but the
name would no longer be permitted on product labels, etc. monograph contains the subsidiary tide 'Codeine Oral
In order to provide continuity, a statement was therefore Solution' and the Definition states that 'Codeine Linctus is
inc1uded in all affected monographs in the British an oral solution containing ... '.
V-A640 Supplementary Chapter II B 2014
5. Standard Terms, as published by the Council of Europe,

are to be used for describing the pharmaceutical form in the
Surnmary of Product Characteristics (SmPC), Patient
Information Leafiet (PIL) and labelling of medicinal products
receiving a European Communiry authorisation. Member
States may use different product names for national
authorisations.
2014 Supplementary Chapter II C V-A641
C. Structures and Nomenclature of Substances of Natural or Semi-synthetic Origin

These notes provide a guide ro the semi-systematic chemical nomenclature of certain groups of natural and semi-synthetic
products. The following literature should be consulted for further information.
1. The definitive rules of the lnternational Union of Pure and Applied Chemistry (lUPAC) for organic substances are
contained in Nomenclature of Organic Chemistry, Section A ro F and H, Pergamon Press, Oxford, 1979.
2. The lUPAC rules for inorganic substances are given in Nomenclature of Inorganic Chemistry, Blackwells, Oxford, 1990.
3. Biochemical Nomenclature and Related Documents, The Biochemical Society for the lnternational Union of Biochemistry
(lUB), 2nd edn. London, 1992. The following topics are included.
Stereochemistry (Nomenclature of Organic Chemistry, Section E)
Natural products and related compounds (Nomenclature of Organic Chemistry, Section F)
Abbreviations and Symbols
Amino acids, peptides, peptide hormones and imrnunoglobulins
Steroids
Carotenoids and tocopherols
Carbohydrates and cyclitols
Vitamins
A. Aminoglycoside Antibiotics
A.1 The aminoglycoside antibiotics are conveniently named by reference ro 2-o-deoxystreptamine, 1, in which name the
configuration and numbering shown are implicit.
A.2 The aminoglycoside antibiotics commonly carry glycosyl radicals on the oxygen aroms attached to C-4 and C-6.
The configuration and numbering shown in 1 should be strictly observed if confusion is to be avoided.
A.3 When one glycosyl radical is linked ro another the names are separated by two locants which indicate the respective
positions involved in this glycosidic union; these locants are enclosed in parentheses and separated by an artow (pointing
from the locant corresponding ro the glycosyl carbon atom to the locant corresponding to the hydroxylic carbon arom
involved). '
B. Cephalosporin Antibiotics
B.1 The cephalosporin antibiotics are conveniently named by reference ro either cephalosporanic acid (2, R = CH 20Ac,
X = H) or cephem-4-carboxylic acid (2, R = X = H).
x-jfJ
O~R
COOH
2 3
B.2 When names are based on cephalosporanic acid or cephem-carboxylic acid the traditional numbering shown in 2 is used.
B.3 Cephalosporanic acid is systematically named as (6R)-3-acetoxymethyl-8-oxo-S-thia-1-azabicyclo[4.2.0]oct-2-ene-2-
carboxylic acid. Compounds that are named systematically use the numbering and orientation shown in 3.
B.4 The cephalosporin antibiotics bear an acylamino group (X) at position 7 (under both numbering systems).
V -A642 Supplementary Chapter 11 C 2014
c. Ergot AIkaloids
C.1 Members of the ergoline group of substances are conveniently named by reference to either ergoline itself, 4, or to
D-Iysergamide, 5. When names are so based the traditionaI numbering shown in 4 is used. In 9,IO-dihydro compounds the
configuration at position-1 O needs to be specified.
4 5 6
C.2 Members of the ergotamine group of substances are conveniently named by reference to ergotaman, 6. Ergotamine itself is
(5' R)-5 '-benzyI-12'-hydroxy-2 '-methyI-18-oxoergotaman-3' ,6 '-dione.
D. Morphines
D .1 Members of the morphine and codeine group of substances have traditionally been named with reference to morphine
itself, 7, using the numbering shown. However, names may be based more conveniently on either morphinan, 8, or
ent-morphinan,9.
7 8 9
D.2 Morphine¡ 7, is (5R, 6S)-4,5-epoxy-9a-methyI-7,8-didehydromorphinan-3,6-diol.

D.3 In certain morphine derivatives, an etheno or ethano bridge is present joining positions 6 and 14 and a hydroxyalkyI side
chain is present at position 7.
E. Penicillin Antibiotics
E.l The penicillin antibiotics are conveniently named by reference to penicillanic acid (10, X = H) ~hen the classicaI
numbering shown is used.
ti ti
X-t---(
_r~ 3111111COOH Me
O H Me
10 11
E.2 Penicillanic acid is systematically named as (2S, 5R)-3,3-dimethyI-7-oxo-4-thia-1-azabicyclo[3.2.0)heptane-2-carboxylic

acid. Compounds that are named systematically use the orientation and numbering shown in 11.
E.3 The penicillin antibiotics are usualIy 6-acylamino penicillanic acid derivatives in which the configuration at position 6 is R.
2014 Supplementary Chapter II C V -A643
F. Polypeptides
F.I The folJowing 3-letter and I-Ietter symbols for amino acids, authorised by the IUPAC-IUB Joint Commission on
Biochemical Nomenclature, are used for representing the sequences of polypeptides:
Alanine Ala A LeL/cine Leu L
Arginine Arg R Lysine Lys K
Asparagine Asn N M ethionine Met M
Aspartic Acid Asp D Phenylalanine Phe F
Cysteine Cys C Pro!ine Pro P
Glutamine Gln Q Saine Ser S
Gluta/l1ic acid Glu E Threonine Thr T
Glycine Gly G Tlypwphan Trp W
Histidine His H Tyrosine Try Y
Isoleucine IIe 1 Valim Val V
F.2 The folJowing symbols recommended by the Joint Commission are also used:
2-Aminohexanoic acid Ahx
Sarcosine Sar
Pyroglutamic acid < Glu
tert-Buwxycarbonyl Boc
F.3 When interpreting sequences of amino-acid residues, the hyphen should be considered as part of the symbol. Its use to
separate the individual residues or radical s may be iIIustrated by the folJowing example:
Gly NHzCHzCOOH
Gly- NHzCHzCO-
- Gly - NHCHzCOOH
- Gly- - NHCHzCO-
and thus,
Gly-Gly-Gly NHzCHzCONHCHzCONHCHzCOOH
The residues are conventionally written with the amino group to the left and the carboxyl group to me right. This is implicit in
the symbolism.
F.4 Where peptide sequences are shown using 3-letter symbols alJ amino acids except glycine have the 'L-' configuration unless
otherwise indicated.
G. Prostaglandins
G.I Members of me prostaglandin group of substances are conveniently named by reference to prostanoic acid, 12, using the
numbering shown. Prostanoic acid may be systematicalJy named as 7-[(1S, 2S)-2-octylcyclopentyl]heptanoic acid.
Dinoprost, 13, may therefore be named as (5Z,12E)-(9S, 11R, 15S)-9, 11, 15-trihydroxyprosta-5,13-dienoic acid.
H H
9
",~COOH
8 \,\ 7 1
"",,~COOH
H
12 13
G.2 A convenient trivial nomenclature exists by which prostaglandins (PG) are c1assified into groups A to F according to me
substitution pattem in me cyclopentane ring (shown below). The subscript numerals 1, 2 and 3 refer to the number of
double bonds found in the side-chains and subscript a refers to the configuration of the C-9 hydroxy group. Thus, 13 is
referred to as PGF 2a .
o o
te <X A B e o E F
V-A644 Supplementary Chapter II C 2014
H. Steroids
H.1 Steroids are drawn and numbered, and the rings lettered, as shown in 14.
24'
26
14 H 15
H.2 Steroids are named with reference to certain basic carbocycles sorne of which are defined in Table 1. When so drawn,
dotted bonds are regarded as lying below the plane of the paper and are designated a, thickened bonds are regarded as
lying aboye the plane of the paper and are designated b and bonds of unknown configuration are shown by a wavy line
and are designated x. .
Table 1
R1 R2 R3
Gonane H H H
Estrane H Me H
Androstane Me Me H
Pregnane Me Me Et
Me~
H
16
H.3 When the hydrogen atom at C-S is present, its configuration is always specified, eg Sa-pregnane, Sb-androstane.
The configuration at centres, 8,9,10, 13, 14 and 17 is assumed to be as shown in 15 unless otherwise specified.
H.4 When inversion of the normal configuration occurs, the positions concerned are specified; and thus 16 is named
Sb,17a-pregnane.
H.5 When inversion occurs at al! of the defined asymmetric centres, the original name is preceded by the italicised prefix ent-.
Racemates are indicated by use of the italicised prefix rae-o
H.6 Further fundamental carbocycles are defined thus:
Table II
Side-chain carbon positions present
Cholane 20 - 24
Cholestane 20 - 27
Ergostane 20 - 27,24 1
In addition to retaining the configuration shown in 15, C-20 has an R-configuration in each carbocycle and C-24 in
ergostane has an S-configuration. However, additional substituents at positions C-17, C-20, C-21 may alter the
R and S propriety descriptions without any change at C-20.
H .7 A large number of therapeutically active steroids bear a carbonyl group at position 3 and unsaturation across
positions 4 and 5. Ring A is often aromatic in estrogens.
2014 Supplementary Chapter II C V -A645
J. T etracyclines
J.1 The tetracycIine antibiotics are conveniently named by reference to tetracycIine itself, 17, (R = H), which may be defined
as (4S,4aS,5aS,6S, 12aS)-4-dimethylamino-1 ,4,4a,5a,6, 11, 12a-octahydro-3,6,10,12, 12a-pentahydroxy-6-methyI-1, 11-
dioxonaphthacene-2-carboxamide.
OH O
CON~
OH
17
J.2 When analogues of tetracycIine are named the stereodescriptors R and S may be subject to change even though the steric
configuration usuaIly remains unchanged. For example, in oxytetracycIine, the hydroxyI group at position 5 imposes
assignment inversions at positions 4a and 5a from Sto R although the steric configuration at these positions remains
unchanged.
Table III
Position
4 4a 5 5a 6 12a
Ch10rtetracycIine S S S S S
Clomocycline S S S S S
Demeclocycline S S S S S
Doxycycline S R S R R S
Meclocycline S R S R S
Methacycline S R S R S
Minocycline S S R S
Oxytetracycline S R S R S S
Tetracycline S S S S S
J.3 When fuIly systematic names based on naphthacene-2-carboxamide are used, the stereodescriptors given in Table In
should be used.
K. Tropanes
K.1 Members of the tropane group of substances are conveniently named by references to tropane itself, 18, when the
numbering and orientation shown are used. Tropane is defined as (8r)-N-methyI-8-azabicyc1o[3.2.1]octane.
H
H Ph
18
O~OH
O
19
V -A646 Supplementary Chapter II C 2014
K.2 Atropine, 19, is (lR,3r,5S,8r) tropan-3-yl (RS)-tropate where the term trapa te represents 3-hydroxy-2-phenylpropionate.
K.3 In the (- )-series the tropoyl side-chain has the (S) configuration, 20.
K.4 In y-tropane compounds, 21, the configuration at position-3 is designated as '3s'.
K.5 In hyoscine and other atropine derivatives bearing a 6,7 -epoxy bridge, the configuration is as shown in 22.
I H Ph (, H Ph
O~OH 'r0~OH
O H O
20 21
Me
o~
22
L. Xanthines
Members of the xanthine group of substances are conveniently named by reference to purine, 23, when the non-systematic
numbering shown is used .
H
N
:~
N
23
2014 Supplementary Chapter III V -A64 7
Organic Chemicals 2 Dr P Holland 6560

Supple~entary Mrs M Barren 6555
Mr A Evans 6565
Chapter III Ms H Corns 6642
Mr JPound 6430
Pharmacopoeial Organisation Pharmaceutical aspects Mrs M Vallender 6562
This Supplementary Chapter provides information that may be Mr S Young 6790
helpful to those wishing to communicate with the British Miss C Pitt 6124
Pharmacopoeia Commission. Radioactive materials Mrs M Barren 6555
Reagents Mr A Evans 6565
Ms H Corns 6642
Surgical materials Mrs M Vallender 6562
A1. Contact Points Unlicensed Medicines Dr F J Swanson 6567
The following table gives an indication of the member of the Miss C Pitt 6124
British Pharmacopoeia Secretariat responsible for particular
Website
areas of the Commission's work. The contact number is
+44 (0)20 3080 followed by the extension listed in the table www.pharmacopoeia.com MrA Gibb 6185
below. Mr J Pound 6430
E-mail (for reference bpcrs@mhra.gsi.gov.uk
substances)
E-mail (for general bpcom@mhra.gsi.gov.uk
enquiries) A2. Expert Advisory Groups
E-mail (for invented inventednames@rnhra.gsi.gov.uk The following table gives an indication of the members of the
names) British Pharmacopoeia Secretariat responsible for particular
Website www.pharmacopoeia.com areas within the remit of the Expert Advisory Groups of the
British Pharmacopoeia Commission. Contact telephone
Editor-in-Chief Mrs M Vallender 6562 numbers are provided in Supplementary Chapter In Al .
Head of AnalyticaI Mr S Young 6790 Expert Class Secretariat
Science Advisory
Group
British Pharrnacopoeia ChemicaI Reference
MC!: Aromatic Synthetics Dr P Holland
Substances (BPCRS)
Medicinal Heterocyc1ic Mrs M Barrett
BPCRS sales Mrs D Myburgh 656 1 Chemicals synthetics
Mr W Jeffries 6564
Alkaloids and
Mr B Delahunty 6144 synthetic analogues
Miss J Paine 6470 MC2: Medicinal Aromatic Synthetics Mr J Pound
BPCRS technical' queries Mr S Young 6790 Chemicals Sulfonamides
Monographs Phenothiazines
General enquiries Mrs D Myburgh 656 1 MC3: Medicinal Steroids and Mr A Evans
Antibiotics Dr F J Swanson 6567 Chemicals pseudosteroids Ms H Corns
Mr Alistair Gibb 6185 Barbiturates
Analytical methods (general) Mr S Young 6790 Carbohydrates
Mr J Pound 6430 Tricyc1ics;
European Pharmacopoeia Mrs M Vallender 6562 benzodiazepines
(general matters)1 Mrs M Barren 6555 Quaternary
Mr M Whaley 6224 ammonium
General monographs Mrs M Vallender 6562 compounds
Miss C Pitt 6124 Vitamins
Herbal medicines Dr P Holland 6560 ABS: Antibiotics Antibacterials Dr F J Swanson
Dr R A Pask-Hughes 6558 Antibiotics Mr A Gibb
Mr M Whaley 6224 HCM: Herbal Crude Drugs Dr P Holland
Inorganic chemicals Mr A Evans 6565 and Homoeopathic Dr R A Pask-
Complementary preparations Hughes
Ms H Corns 6642 Medicines
Microbiological aspects MrM Whaley 6224 Traditional Herbal Mr M Whaley
Dr R A Pask-Hughes 6558 Materials
New monographs Ms H Corns 6642 NOM: Nomenc1ature Dr P Holland
Nomenclature Invented N ames Mr A Evans
(general matters) 1 Mr Alistair Gibb 6185
Nomenclature Dr P Holland 6560 Ms H Corns
Mr A Evans 6565
Ms H Corns 6642
1 For specijic mauers see under relevant subjecr entry 2 See !able in Supplementary Chapeer 111 A2
V-A648 Supplementary Chapter III B 2014
PCY: Phannacy Phannaceutical Mrs M Vallender

aspects Miss C Pitt
B. Monograph Development: Mechanism
The following Supplementary Chapter provides an outline of the
General
mechanism by which monographs are selected and developed for
rnonographs
inclusion in the British Pharmacopoeiao
Phannaceutical
The British Phannacopoeia Commission will not usually
techniques
develop rnonographs for drug substances or excipientso These
Dosage forrn will usually be elaborated by the European Phannacopoeia
rnonographs Comrnissiono
(general)
The British Phannacopoeia Cornmission will consider a
Unlicensed General Dr F J Swanson
rnonograph for inclusion in the British Phannacopoeia in the
Medicines rnonographs (ULM) Miss C Pitt
following circumstances:
Forrnulated
lo The fonnulation is widely used (for example: products
Preparations
in the top 500 list of prescribed iterns; products in the
top 100 list of items used in hospitals; Phannacy (P) and
General Sale List (GSL) products that are widely used,
taking into account seasonal fiuctuations; veterinary
products that are widely used or where a need is
idemified) o
BP Commis$ion
BP Laborato.ry
Practical assessment
Method development
Expert Advisory Group
Technical advice
Approve final draft
Final monograph BP Secretariat

. ~to .........
Invite proposªls
BP Commission Dra.ft monograph
Draft monographs Feedback

2014 Supplementary Chapter III C V -A649
2. The i=ovator product is approaching or past its patent described in Supplementary Chapter 1 and to other
expiry date (monographs will usually only be prepared in information provided in the Supplementary Chapters of the
the two years preceding patent expiry. However there Pharmacopoeia.
may be circumstances where it is justified to prepare a 1. DefinitionlCharacteristics Brief description of physical
monograph at an earlier stage. These will be considered form of material; whether hygroscopic. It would be helpful if
by the British Pharmacopoeia Commission individually). information concerning polymorphism could be provided (see
3. There is a particular need based on the therapeutic Supplementary Chapter 1 B).
category and/or the importance of the material 2. Solubility Solubility in water, alcohol and at least one
concemed; the latter being particularly relevant to small common organic solvent, expressed quantitatively or using
patient populations. defined BP terms.
4. Unlicensed products that are produced to meet 3. Identification Qualitative tests capable of unequivocally
particular patient needs (generally "specials" establishing the identity of the substance. Infrared
manufacture or extemporaneous preparation) (see also spectrometry is preferred, or if this is not possible, 2 or 3
Supplementary Chapter V A). identification tests to identify different characteristics of the
5. The productlpreparation is a widely used traditional substance may be used. A test for the counter ion should be
herbal medicine. included where appropriate.
6. The legal classification of the product has been changed 4. Impurities (see also Supplementary Chapter 1 A) Both
from POM (prescription only medicine) to P or from P related substances and any other impurities that may be
to GSL. present in the substance as a result of the method of
7. The product falls within a "family" of product manufacture or from degradation on storage. It would be
presentations, of whicll there are already published helpful to know the nature of such impurities, the reason for
monographs and/or monographs on the work their presence (for example, by-product of synthesis,
programme. hydrolysis product, etc.), the amounts that may be
8. Drug substances or excipients which are not on the encountered in material prepared under conditions of Good
European Pharmacopoeia work programme, but for Manufacturing Practice and the manner in which the
which there is a specific UK need. proportions may vary on storage. An indication of the toxicity
of any impurities in relation to that of the substance itself
9. A request is received from the Competent Authority and methods for their detection and control would enable the
[Medicines and Healthcare Products Regulatory Agency Committee preparing the monograph to decide whether
(MHRA)].
control tests are necessary and, if so, the methods and limits
10. A request is received from a manufacturer for one of to be applied. In many cases, it is possible to limit impurities
their own products. in chromatographic tests using a dilute solution of the
11. Support for relevant EC directives. substance being examined, and this is the preferred
12. A request is received from official bodies [such as the approach, wherever possible. In other cases, for example,
World Health Organization (WHO)]. where the response factor of an impurity is significantly
different from that of the substance (outside the range 0.8 to
13. Other circumstances considered on a case-by-case basis.
1.2), or where a stringent limit is necessary for toxicity
It should be noted that compliance with any of the aboye reasons, a reference material is required (see paragraph 11
criteria will not necessarily mean that a monograph wili be below and Supplementary Chapter III E).
included in the British Pharmacopoeia. The B~itish
Impurities other than related substances that might require
Pharmacopoeia Commission may decide not to elaborate a
control include inorganic impurities, heavy metals (especially
monograph for a number of reasons, including a lack of
for chronically administered or high-dosage materials) and
interest fram stakeholders, resource limitations or other
residues of solvents and reagents used during synthesis and
circumstances, decided on a case-by-case basis.
purification. Non-specific purity tests such as light
The diagram provides a simplified, schematic representation of the absorption, specific optical rotation and sulfated ash should
development of a monograph for a medicinal substance 01' an also be considered.
associated formulated pl'eparation.
5. Transparency In keeping with BP policy on monograph
transparency a suitable statement will be added to
appropriate new monographs for medicinal substances giving
C. Monograph Development: Guidance the identities of impurities known to be limited by the
specifications. It is to be emphasised that such statements are
to Manufacturers not intended to be exclusive and other, unnamed impurities
Bulk drug substances may also be limited. Manufacturers are requested to provide
information for such statements (see paragraph 4 aboye) .
Each monograph, taken as a whole, should provide a reliable
basis for making an independent judgement as to the quality 6. Assay Method and proposed limits calculated with
of the substance in the interests of the protection of the reference to the anhydrous, dried or solvent-free material as
public. General guidance as to the types of test required and appropriate. For bulk drug substances, it has been BP policy
the level of control considered appropriate can be obtained generally to use a robust and precise method of assay (such as
by reference to current BP monographs for similar chemical titration) rather than a specific, but sometimes less precise,
entities where such are available. In general any monograph stability-indicating method (such as liquid chromatography).
for a bulk drug substance should include the features listed Wherever possible, control of potential impurities is provided
below. separately by means of specific impurity tests (see paragraph
4 aboye). It is appreciated, however, that a manufacturer may
Attention is drawn to the General Notices of the
use, and therefore propose, a chromatographic method for
Pharmacopoeia, to the general methods described in the
both related substances and assay. In such circumstances,
Appendices, to the basis of pharmacopoeial requirements as
V-A650 Supplementary Chapter III C 2014
each case is judged on its merits on the basis of the data Fonnulated preparations
provided, which must relate to validated methods . Such Each monograph, taken as a whole, should provide a reliable
methods normany require the establishment of a reference basis for making an independent judgement as ro the quality
material of the substance with a deelared content (se e of the preparation in the interests of the protection of the
paragraph 11 below and Supplementary Chapter III E). pub lic. General guidance as to the types of test required and
Adequate means of demonstrating system suitability will need the level of control considered appropriate can be obtained
ro be ineluded in the monograph so that the analyst has an by reference ro current BP monographs for the same dosage
assurance that the results are accurate. As stated aboye, each form of similar chemical entities where such are available.
monograph, taken as a whole, should provide a reliable basis Reference should also be made to any general monograph for
for making an independent judgement as to the quality of the the dosage form in question for general requirements and any
substance. exceptions to, or modificaríons of, those requirements noted.
7. Other tests A test for water or for loss on drying is In general any monograph for a formulated preparation
usuany required. should inelude the features listed below.
8. Storage Any special srorage conditions such as protection Attention is drawn ro the General Notices of the
from light. Pharmacopoeia, to the general methods described in the
9. Labelling Any speciallabening statements (see Appendices, ro the basis of pharmacopoeial requirements as
Supplementary Chapter 1 G). described in the Introduction and Supplementary Chapter 1
10. Preparations 'Pharmaceutical dosage forms normany and to other information provided in the Supplementary
available and information on dose. Chapters of the Pharmacopoeia.
11 . Samp1es A quantity of the material sufficient to carry l . Definition/Description A definiríon of the prepararíon in
out in duplicate an the tests ' and the assay in the proposed terms of the active ingredientes) together with informaríon on
specification should be supplied (lO gis usuany suitable). its presentaríon.
This sample shoulci be taken from a typical production For sterile preparations (parenteral, ophthalmic and others)
batch, that is, it should not be speciany purified. In addition this should inelude information on the nature of the vehiele;
appropriate amounts of possible impurities should be the nature of any additives (eg antimicrobial preservatives,
supplied (0.1 to 0.5 gis usuany suitable). buffers) present; the method of sterilisation. In addition, for
Many monographsrequire the use of one or more reference parenteral preparations informaríon should be provided on
substances (BPCRS); these are established by the Laborarory whether it is a solution, a suspension, a dry powder or a
before publication of the monograph (see Supplementary concentrate for dilution.
Chapter In E). If a reference substance for assay purposes is For topical semi-solid preparations this should inelude
required (see paragraph 6 aboye) or for impurity control (see information on the type of basis (water-in-oil, oil-in-water,
paragraph 4 aboye), pIe ase indica te if adequate supplies will etc) and the parrícle size of the active ingredient, if
be available. About 50 g of material is required to establish a significant.
reference material for an assay standard and about 10 g of a For tablets this should inelude whether or not they are
named impurity (see also Supplementary Chapter III E). If it coated. Where tablets are coated, the reason for coaríng
is perceived that an ongoing supply of sufficient quantities of should be provided.
materiales) will not be possible, this should be drawn ro the Information concerning polymorphism should be ineluded
attention of the BP Sécretariat so that an altemative where relevant (se e Supplementary Chapter 1 B).
approach may be considered.
2. Content statement Proposed limits as a percentage of
When sending samples, Material Safety Data Sheets that the stated content of the active ingredient in the terms
comply with COSHH Regulations should be supplied for an deelared on the label (se e Supplementary Chapter 1 F).
materials ineluding impurities, so that Pharmacopoeia staff
The purpose of the assay in prepararíon monographs is ro
are aware of possible hazards when handling these materials.
determine whether the content of the active ingredient is
12. Supporting data Appropriate and relevant validation within acceptable limits of the labened elaim and the limits
data relating ro the proposed analytical procedures and are therefore of necessity stated in terms of the moiety declared
methodology should be provided (see Supplementary on the {abe!. That is, the same method of expression is used in
Chapter In F). In particular, data to demonstrate the the content statement as under the headings Assay and
stability indicating nature of methods (i.e. forced degradation Labelling. The preferred means of expression is in terms of
studies) and information identifying both synthetic impurities the therapeuticany active part of the molecule. It should be
and degradation products should be provided. Additionany, noted that the mode of expressions chosen for the assay
appropriate and relevant batch and stability data to support limits in the monograph for the bulk drug substance in no
the proposed specifications should be provided. This way circumscribes that which may be used in the monograph
informarían win be kept confidential ro the BP Secretariat for the formulation.
and Laboratory and ro the relevant Expert Advisory Group (s)
3. Identification Identificaríon tests should be based on
of the British Pharmacopoeia Commission.
those for the parent drug substance where applicable, with
13. Advice The BP Secretariat is pleased to provide advice details of any necessary preliminary treatment such as
ro manufacturers on the nature and extent of data required. extraction. Infrared spectrometry is preferred. A single
Ir is appreciated that for some older products informaríon on liquid-chromatographic method for Identification, control of
the identity of impurities ancl/or validaríon data for methods impurities, and Assay should be supplemented by an
is not as substantial as that which is required for new additional test for identification.
chemical entities and advice in these cases is available. A list
4. Impurities [As under Bulk drug substances] Addiríonal
of contact points in the Secretariat is to be found in
informaríon on any impurities arising on manufacture or
Supplementary Chapter III A.
srorage of the dosage formo
2014 Supplementary Chapter III D V-A651
The tests applied to the bulk drug substance, including those When sending samples, Material Safety Data Sheets that
for impurities arising in manufacrure of the bulk drug comply with COSHH Regulations should be supplied for all
substance, should be applied, wherever possible - with any materials including impurities, so that Pharmacopoeia staff
necessary modification - in order to demonstrate that are aware of possible hazards when handling these materials.
material of pharmacopoeial quality has been used in making 12. Supporting data Appropriate and relevant validation
the formulation. data relating to the proposed analytical procedures and
5. Transparency In keeping with BP policy on monograph methodology should be provided (see Supplementary
transparency a suitable statement will be added to Chapter II! F). In particular, data to demonstrate the
appropriate new monographs for formulated preparations stability indicating nature of methods (i.e. forced degradation
giving the identities of impurities known to be limited by the studies) and information identifying both synthetic impurities
specifications. It is to be emphasised that su eh statements are and degradation products should be provided. Additionally,
not intended to be exclusive and other, unnamed impurities appropriate and relevant batch and stability data to support
may also be Iimited. Manufacrurers are requested to provide the proposed specifications should be provided. This
information for such statements (see paragraph 4 aboye). information will be kept confidential to the BP Secretariat
6. Assay The method of assay will not necessarily be that and Laboratory and to the relevant Expert Advisory Group(s)
used for the bulk drug substance. For formulated of the British Pharmacopoeia Commission.
preparations a specific, stability-indicating method is 13. Advice The BP Secretariat is pleased to provide advice
preferred. Such methods normally require the establishment to manufacturers on the nature and extent of data required.
of a reference material of the substance with a declared Ir is appreciated that for sorne older products information on
content (see paragraph 11 below and Supplementary Chapter the identity of impurities andlor validation data for methods
III E). is not as substantial as that which is required for new
7. Other tests Tests such as pH and c1arity and colour of chemical entities and advice in these cases is available. A Iist
solution may be necessary depending on the type of dosage of contact points in the Secretariat is to be found in
form o In addition, for single-dose preparations a test for Supplementary Chapter III A.
uniformity of content and, in the case of solid dosage forms,
a dissolution test may be required (se e Supplementary
Chapter I E).
8. Storage Any special storage conditions/containers. D. Monograph Development: Methods of
9. Labelling ~ny speciallabelling statements (see
Supplementary Chapter I G).
Analysis
1. This Supplementary Chapter concems the methods
PIe ase pro vide sample labels, outer packages, leafiets and a
described in the Pharmacopoeia for the analysis of medicinal
copy of the relevant Summary of Product Characteristics
and pharmaceutical substances, pharrnaceutical dosage forms
(SmPC) or data shee~.
and other articles. Ir provides information on the
10. Strengths available/Dose While no longer included in development, validation and use of pharmacopoeial methods
the published monograph, this information is of assistance so that users of the Pharmacopoeia may understand the
during monograph development. purpose and limitations of a monograph and to guide
11. Samples A quantity of the formulation sufficient to carry manufacrurers and other users when participating in the
out in duplicate all the tests (including those under development of new monographs and the revision of existing
paragraph 7) and the assay in th~ proposed specification monographs.
should be supplied. Samples should be taken from a typical 2. For further information regarding the elaboration of
production batch. European Pharmacopoeia monographs, users may also
The following suggested quantities are provided as a rough consult the " Technical guide for the elaboration of
guide of the order of sample size for each strength of monographs", which is published by the European
different dosage forms: Pharmacopoeia .
Solid single dose formulations 100 units 3. The Chapter does not set out the nature and extent of
(Tablets, Capsules, etc) validation required in particular instances. Guidelines on
Liquid formulations such matters with respect to product registration are available
from other sources such as the Intemational Conference on
i) Topical and Oral formulations 100 mL
Harmonisation (ICH) and are published as guideline
ii) Parenteral formulations 50 mL
Q2 (R1 ), "Validation of Analytical Procedures: Text and
Semi-solid topical formulations 50 to 100 g
Methodology". The terms used, and illustrations of the data
In addition appropriate amounts of possible impurities requirements to demonstrate that a method satisfies the
(arising from synthesis or degradation) should be supplied definitions, are provided in Supplementary Chapter III F.
(approximately 200 to 500 mg, unless the impurity is
required for use as a reference standard, see below) Method origin
Many monographs require the use of one or more reference 3. Proposals for new or revised methods for inclusion are
substances (BPCRS); these are established by the Laboratory often provided by manufacturers and other users of the
before publication of the monograph (see Supplementary Pharmacopoeia either in response to a request from the
Chapter lIT E). If a reference substance is required for assay Secretariat or Laboratory or, for revised methods, when a
purposes (see paragraph 6 aboye), or for impuriry control published method has been found to be unsuitable for any
(see paragraph 4 under bulk drug substances), please indicate reason. Amethod may become unsuitable when, for
if adequate supplies will be available . About 50 g of material example, a reagent or piece of appararus is no longer readily
is required to establish a reference material for an assay available, knowledge of the material has increased, regulatory
standard and about 10 g of a named impurity (see also requirements have altered or a more specific or sensitive test
Supplementary Chapter III E) . has been developed.
V-A652 Supplementary Chapter 111 D 2014
4. Any method that is to be considered for inelusion in a 6. The evaluation of methods is carried out by the
monograph has to be suitable for phannacopoeial purposes Commission's Expert Advisory Groups and Panels of
and, wherever possible, be accompanied by appropriate Experts, the Secretariat and the Laboratory; practical
supporting data. evaluation is ineluded in many cases. If necessary, more
4.1 Guidance on the suitability of any method for inelusion extensive practical work is carried out in consultation with
in the Phannacopoeia may be obtained by reference to the proposer of the method. Such practical work may be
the General Notices (in particular, those dealing with necessary, for example, where a proposed method is not
official standard s, excipients, identification and assays direcdy applicable to other sources of the material or
and tests) and Supplementary Chapter 1 conceming the preparation or is shown to be insufficiently robust for
basis of phannacopoeial requirements. Each monograph, phannacopoeial use.
taken as a whole, should provide a reliable basis for 7. After initial evaluation, the method is drafted in the style
making an independent judgement as to the quality of of the Phannacopoeia and those known to have an interest in
an artiele in the interests of the protection of the publico the material or preparation are invited to comment.
4.2 A phannacopoeial monograph applies throughout the If necessary, further modifications may be made to the
shelf-life of a formulated preparation or throughout the method and the consultation process repeated before the
period of use of a medicinal or phannaceutical substance method is published. A diagrammatic representation of the
and the monograpli. when taken as a whole must be process of monograph elaboration is provided as
stability indicating and must be able to assure the quality Supplementary Chapter III B.
of the substance throughout its elaimed shelf life.
Methods for inelusion in the Phannacopoeia should Published methods
therefore have been shown to be stability indicating 8. The user can expect that published methods:
where appropriate by means of forcéd degradation are suitable for the purpose for which they are described
studies. in the Phannacopoeia, have been evaluated and, where
4.3 Other than in exceptional circumstances, methods necessary, modified as described in the preceding
shouId not specify the use of apparatus that is not widely section,
available in reasonably equipped laboratories nor should have been shown to be adequately validated, as
they require extensive additional training of laboratory appropriate to the type of test, and inelude tests to
staff. Reagents and reference material s required for a demonstrate the continuing suitability of the method,
proposed phannacopoeial method should be generally where necessary,
available from the common sources of supply in the are described in sufficient detail that a competent analyst
United Kingdom or a manufacrurer should be able to can perfonn the test using readily available apparatus
supply sufficient quantity for use as a British and reagents and that any necessary reference materials
Phannacopoeia Chemical Reference Substance are available, will be reviewed and revised when
(BPCRS). The method should be described in sufficient experience shows this to be necessary.
detail that a competent analyst is able to repeat it.
9. The user cannot, however, assume that a method forming
4.4 Methods proposed by manufacrurers shouId have been part of a phannacopoeial monograph will have been applied
adequately validated in accordance with appropriate to all sources of a raw material or to aH formulations of a
guidelines (paragraph 2 refers) . Exceptionally, full dosage form currently available. The user is responsible for
validation data may not be available in sorne confinning that the method is applicable to the particular
circumstances. For example, not aH contributors to the material being exarnined. It is essential that a manufacturer
Phannacopoeia are able to demonstrate the specificity of carries out sufficient checks to demonstrate that, for example,
a test method for application to a monograph for a impurities arising from a new route of synthesis are
formulated preparation since the narure of the excipients controHed by the methods described in the monograph or
may be unknown; in this case a note of the extent of any that the excipients in a formulated preparation do not
validation carried out, with its limitations, is helpful. interfere with any of the tests in a monograph. It should be
4.5 It is of particular importance that method proposals noted that an artiele cannot elaim to be of phannacopoeial
inelude a robust and reliable identity testes) for the quality unless it can be shown to comply with aH of the tests
active component in a substance or formulated specified in a monograph (see General Notice on Official
preparation. Infrared spectrophotometry is preferred. Standards) .
4.6 It is also of particular importance that any
phannacopoeial method is robust and reproducible. Data Feedback
that demonstrate the transferability of a method are, The British Phannacopoeia Comrnission welcomes
therefore, especiaHy helpful. constructive comments from users on the tests of the
Phannacopoeia and it is through such feedback that revision
Method elaboration
of the tests is initiated . A list of contact points in the
5. AH proposals for new and revised methods for publication Secretariat is to be found in Supplementary Chapter III A.
are carefuHy examined. The narure and extent of the Altematively, please complete the feedback form ineluded in
evaluation is detennined by a number of factors ineluding the the publication and send it to the British Phannacopoeia
foIlowing: Commission Office, 5th Floor, 151 Buckingham Palace Road,
- whether the pro pos al is for a new monograph or for London, SWl W 9SZ, marked for the attention of the
revision of an existing monograph, Editor-in-Chief.
- extent of validation and batch data available,
- how many specifications and/or samples are available for
examination,
- complexity of the propose(i method.
2014 Supplementary Chapter III F V-A653
replacement stocks. As a service to analysts, reference

E. British Pharmacopoeia Chemical substances for monographs omitted from the current edition
Reference Substances (BPCRS) of the Pharmacopoeia are normally retained for about five
l. The current list of BPCRS may be found on the BP's years from the date of publication of the current edirion
website at www.phannacopoeia.com. The quantity of material unless the material becomes unsatisfactory or the supply is
supplied is sufficient to carry out in duplicate each of the exhausted before that date.
tests in which it is used in any one monograph. 9. Demand is monitored and any significant increase in
demand for a reference substance for an omitted monograph
Requirement is noted. If such increased demand appears to stem from a
2. The establishment of a new BPCRS is based on scientific renewed interest in the medicinal or pharmaceutical product,
necessity following advice. from the British Pharmacopoeia consideration may be given to reinstating the monograph in
Commission's Expert Advisory Groups. A reference the Pharmacopoeia.
substance for the medicinal substance is usually required for
specific, stability-indicating assays and may also be required
for identificatio'n purposes. A reference substance for an
impurity is required when control using a dilute solution of F. Validation of Analytical Procedures
the substance being examined is not possible or is
undesirable. Such cases inc1ude those where the response Introduction
factor of the impurity is significantly different from that of Validation of an analytical procedure is performed in order to
the substance, or where it is necessary to limit a named demonstrate that the procedure is suitable for its intended
impurity (see Supplementary Chapter I A). use. Validation is performed in order to show that the
The following criteria are taken into account when resultes) generated by a particular analytical procedure are
determining whether a new BPCRS is required. reliable and accurate.
Availability of the material A commercially available The principies and practices of validation of analytical
reagent is specified wherever it is found to be suitable and procedures are covered by the Intemational Conference on
justified. Harmonisation (ICH), are published as guideline Q2(Rl),
AnalyticaI convenience Wherever possible, either the "Validation of Analytical Procedures: Text and
same EPCRS or the same BPCRS is specified throughout Methodology" and are available from www.ich.org. A full
any one monograph. discussion of the terms and methodology applicable to
validation of analytical procedures is provided in the ICH
Establishment documents.
3. Manufacturers who have contributed to the development
Types of procedures to be validated
of the monograph(s) are asked to supply material for use as
reference substances. Normally, about 50 g is required for an The objective of validation of an analytical procedure is to
Assay standard and about 10 g for an impurity. In sorne demonstrate that it is suitable for its intended purpose.
cases it is possible to use a quantity of the medicinal A tabular summation of the characteristics applicable to
substance admixed with smaller quantities of the impurities identification, control of impurities and assay procedures is
as an impurity reference standard. It would be helpful if inc1uded.
manufacturers c;ould indicate the likely availability of - signifies that this characteristic is not normally evaluated
reference materials at an early stage during monograph + signifies that this characteristic is normally evaluated
development. (1) in cases where reproducibility has been performed,
4. The material s are tested by the Laboratory to confirm intermediate precision is not needed
their suitability for the intended purpose and are made (2) lack of specificity of one analytical procedure could be
available not later than the effective date of the monograph in compensated by other supporting analytical procedure(s)
which they are used.
(3) may be needed in sorne cases
Monitoring and replacement Specificity
5. AII substances used for quantitative analyses are re-tested Specificity is the ability to assess unequivocally the analyte in
every three years and material s used for qualitative analysis the presence of components that may be expected to be
every five years unless experience has shown that more presento Typically these might inc1ude impurities, degradants,
frequent testing is necessary. matrix, etc.
6. When a BPCRS is due for replacement (because of a fall Specificity may often be expressed as the degree of bias of
in quality or exhaustion), a review of the use of the material test results obtained by analysis of samples containing added
and the number of units supplied is carried out. impurities, degradation products, related chemical
compounds, or placebo ingredients when compared to test
Continued availability results without added substances.
7. When a monograph is omitted from the Pharmacopoeia Specificity is usually demonstrated by measuring the response
the last published monograph remains the legal standard. of the sample matrix and any expected or known species (for
As the usual reason for omission is low and dec1ining use or example excipients, impurities or degradation products).
withdrawal of the material from the market, demand for any It would normally be expected that no significant response
associated reference substances from within the United would be obtained that interferes with the measurement of
Kingdom is normally low. the analyte(s). However it is not always possible that an
8. It is not possible to maintain reference substances for analytical procedure is specific for a particular analyte. In this
omitted material s indefinitely and, for material s no longer instance a combination of two or more analytical procedures
marketed in the United Kingdom, it is difficult to obtain may be necessary to achieve the required discrimination.
V-A654 Supplementary Chapter III F 2014
Type of analytical IDENTIFICATION TESTING FOR IMPURITIES ASSAY

procedure - dissolution
(measurement only)
- contenUpotency
characteristics quantitat. limit
Accuracy - + - +
Precision
Repeatability - + - +
Interm . Precision - + (1) - + (1)
Specificity (2) + + + +
Detection Limit - - (3) + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
Linearity Usually a minimum of three determinations at each of three

The linearity of an analytical procedure is its ability (within a concentrations across the intended range is recommended.
given range) to obtain test results that are directly Accuracy may also be demonstrated by the method of
proportional 10 the concentration (amount) of analyte in the standard additions, or by cross-correlation of results with a
sample. second, independent, procedure. Accuracy may be inferred
Linearity is usually demonstrated by visual inspection of a once precision, linearity and specificity have been established.
plot of signals as a function of analyte concentration or Precision
conten!. If there is a linear relationship, test results should be The precision of an analytical procedure expresses the
evaluated by appropriate statistical methods, for example, by
closeness of agreement (degree of scatter) between a series of
ca1culation of a regression line by the method of least
measurements obtained from multip1e sampling of the same
squares. In some cases, to obtain linearity between assays and
homogeneous sample under the prescribed conditions.
sample concentrations, the test data may need 10 be
Precision may be considered at three levels: repeatability,
subjected 10 a mathematical transformation prior to the
intermediate precision and reproducibility.
regression analysis. Data from the regression line itseIf may
be helpful to provide mathematical estima tes of the degree of Precision should be investigated using homogeneous,
linearity. authentic samples. However, if it is not possible to obtain a
homogeneous sample it may be investigated using artificially
The correIation coefficient, y-intercept, slope of the
prepared samples or a sample solution.
regression line and residual sum of squares should be
ca1culated. A plot of the data should be included. The precision of an analytical procedure is usually expressed
In addition, an analysis of the deviation of the actual data as the variance, standard deviation or coefficient of variation
points from the regression line may also be helpful for of a series of measurements.
evaluating linearity. REPEATABILlTY (INTRA-ASSAY PRECISION)
A minimum of five concentrations is recommended. Other Repeatability express es the precision under the same
approaches should be justified. operating conditions over a short interval of time.
Repeatability is also termed intra-assay precision.
Accuracy
Repeatability is usually demonstrated by repeated
The accuracy of an analytical procedure expresses the
measurements of a single sample (e.g. use of the analytical
closeness of agreement between the value which is accepted
procedure within a laboratory over a short period of time
either as a conventional tme value or an accepted reference using the same analyst with the same equipment).
value and the value found. This is sometimes termed
A minimum of three determinations at each of three
tmeness.
concentrations across the intended range, or a minimum of
Accuracy should be established across the specified range of six determinations at the test concentration is recommended.
the analytical procedure.
INTERMEDIATE PRECISION
Accuracy is usually demonstrated by adding known amounts Intermediate precision expresses within-Iaboratory variations:
of analyte(s) to the sample matrix and determining the different days, different analysts or equipment, etc.
measured result using the analytical procedure. The recovery
of measured agains t actual amounts is then ca1culated.
2014 Supplementary Chapter III F V-A655
Intermediate precision is usually demonstrated by repeated For detem1ination of impurities the range is usually not less
measurements of the sample used in the repeatability than the reporting limit of the impurity to 120% of the
experiment within the same laboratory. Usually the specification.
repeatability experiment is repeated on the same sample by a For dissolution testing the range is usually +/- 20% over the
different analyst, on a different day, using different expected concentrations.
equipment if possible. Range is usually demonstrated by confirming that the
REPRODUClBILlTY analytical procedure provides an acceptable degree of
Reproducibility expresses the precision between laboratories linearity, accuracy and precision when applied to samples
(collaborative studies, usually applied to standardisation of containing amounts of analyte within or at the extremes of
methodology) . the specified range.
Reproducibility is usually demonstrated by means of an Systern Suitability Testing
inter-Iaboratory tria!.
System suitability testing is an integral part of many
Detection Lirnit analytical procedures. The tests are based on the concept
The detection limit of an analytical procedure is the lowest that the equipment, electronics, analytical operations and
concentration of analyte in a sample that can be detected but samples to be analyzed constitute an integral system that can
not necessarily quantitated as an exact value. be evaluated as such. System suitability test parameters to be
The detection limit is usually expressed as the concentration established for a particular procedure depend on the type of
of analyte (e.g., percentage, parts per billion) in the sample. procedure being validated.
The detection limit is usually demonstrated by measuring low Criteria for assessing the suitability of chromatographic
concentrations of the analyte and showing that a response is systems are described in the chapter on Chromatographic
separation techniques (Appendix nI (Ph. Eur. merhod
obtained.
2.2.46)). The extent to which adjustments of parameters of
Quantitation Lirnit the chromatographic system can be made to satisfy the
The quantitation limit of an individual analytical procedure is criteria of system suitability are also given in this chapter.
the lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and
accuracy. The quantitation limit is a parameter of
quantitative assays for low levels of compounds in sample
matrices, and is used particularly for the determination of
impurities ancl/or degradation products.
It is usually expressed as the concentration of analyte
(e.g. percentage, parts per billion) in the sample.
The quantitation limit is usually demonstrated by measuring
low concentrations of the analyte and showing that a
repeatable response is obtained.
Robustness
The robustness of an analytical procedure is a measure of its
capacity to remain unaffected by small but deliberate
variations in method parameters and provides an indication
of its reliability during normal usage.
Robustness is usually demonstrated by making small
deliberate changes to one of the operating parameters of the
method, analysing samples and comparing the results to
those obtained using the prescribed method.
Range
The range of an analytical method is the interval between the
upper and lower concentration (amounts) of analyte
(including these concentrations) for which it has been
demonstrated that the analy~ical procedure has a suitable
level of precision, accuracy and linearity.
The specified range is normally derived from linearity studies
and depends on the intended application of the procedure.
It is established by confirming that the analytical procedure
provides an acceptable degree of linearity, accuracy and
precision when applied to samples containing amounts of
analyte within or at the extremes of the specified range of the
analytical procedure.
For assays the range is usually not les s than 80 to 120% of
the test concentration.
For determination of content uniformity the range is usually
not less than 70 to 130% of the test concentration.
V-A656 Supplementary Chapter IV 2014
The United Kingdom delegation to the European

Supplementary Pharmacopoeia Commission up to 1 January 2014 was as
follows:
Chapter IV Dr S Atkinson
Mr V Fenton-May
Alternate members of the United Kingdom delegation up to
European Pharmacopoeia 1 January 2014 was as follows:
This Supplementary Chapter pro vides infomzation conceming the
Professor A Davidson
European Phannacopoeia.
Dr R Horder
Mrs M Vallender
A. Membership of the European

UK Membership of Groups of Experts of the
Pharmacopoeia Commission European Pharmacopoeia Commission
In addition to the United Kingdom, the Member States party The United Kingdom is represented in alI of the ma;or
to the Convention on the Elaboration of a European Groups of Experts of the European Pharmacopoeia
Pharmacopoeia are as follows: Austria, Belgium, Bosnia and Commission. The VK membership of the Groups on
Herzegovina, Bulgaria, Croatia, Cyprus, The Czech 1 January 2013 is as listed in the table below.
Republic, Denmark, Estonia, Finland, France, Germany,
Greece, Hungary, !celand, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Montenegro, The Netherlands,
Norway, Poland, Portugal, Romania, Serbia, Slovakia,
Slovenia, Spain, Sweden, Switzerland, The Former Yugoslav
Republic of Macedonia, Turkey and Ukraine. The European
Union is also party to the Convention.
Observers to the European Pharmacopoeia Commission at
the time of publication are: Albania, A1geria, Argentina,
Armenia, Australia, Belarus, Brazil, Canada, China, Georgia,
Israel, Kazakhstan, Madagascar, Malaysia, Moldova,
Morocco, Republic of Guinea, The Russian Federation,
Senegal, Singapore, Syria, Tunisia, the United States of
America and the World Health Organization.
1 Microbiology Mr V Fenton-May
6 Biological Substances Dr A F Bristow
6B Human Blood and Blood Products Dr A R Hubbard
7 Antibiotics Mr B White
9G Medicinal Gases Dr M G Lee (Chairman) Mr P
Henrys
lOA Organic Chemistry (Synthetic Products) Mr D Malpas
10B Organic Chemistry (Synthetic Products) Mr S Arkle
10C Organic Chemistry (Synthetic Products) Mr AJ Caws
10D Organic Chemistry (Synthetic Products) Mr C T Goddard
11 Organic Chemistry (Natural Products) Professor A G Davidson
(Chaimzan)
Mr M Tubby
12 Dosage Forms and Methods Dr R Horder
13A Phytochemistry A Dr K HelliwelI
13B Phytochemistry B Dr K HelliwelI (Chairman)
13H Fatty Oils and Derivatives Dr R Cawthorne Dr M Evans
(Specialist)
14 Radioactive Compounds Dr R D Pickett
15 Sera and Vaccines Dr D Sesardic Dr S Schepelmaun
(Specialist)
15V Veterinary Sera and Vaccines Dr A-M Brady
16 Plastic Containers for Pharmaceutical Use DrKAllen
P4 Procedure 4 Mr S Young
2014 Supplementary Chapter IV B V-A657
UK Membership ofWorking Parties ofthe

European Pharmacopoeia Commission
B. Dates of Implementation
Under the 1964 Convention 1 on the Elaboration of a
The UK membership of the Working Panies on 1 January
European Pharmacopoeia the standards of the European
2013 is as follows.
Pharmacopoeia are required to take preceden ce over the
Alkyl Mesilates Professor J M Midgley standards of the national pharmacopoeias of the contracting
(Chairman) parties, thus ensuring a common standard.
Allergens Dr A Cook
In addition to the United Kingdom the countries pany 10 the
Bacterial Endotoxin Test Dr L Findlay
Convention are: Austria, Belgium, Bosnia and Herzegovina,
Botulinum Toxin Test Dr D Sesardic
Bulgaria, Croatia, Cyprus, The Czech Republic, Denmark,
Cell Therapy Products Dr M O'Kane
Estonia, Finland, France, Germany, Greece, Hungary,
Chromatographic Separation Techniques Mr S Young
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg,
Dialysis Solutions Dr M GLee
Malta, Montenegro, The Netherlands, Norway, Poland,
(Chairman)
Portugal, Romania, Serbia, Slovakia, Slovenia, Spain,
Extracts Dr K Helliwell
Sweden, Switzerland, The Former Yugoslav Republic of
(Chairman)
Macedonia, Turkey and Ukraine. The European Union is
Dr L Anderson
also pany 10 the Convention.
Dr M Pires
Finished product monographs Dr S Atkinson The 8th Edition, published in July 2013, carne into force on
Functionaliry Related Characteristics Mr C Mroz 1 January 2014 and will be amended by Supplements 8.1 10
Gene Therapy Products Dr E Pollitt 8.8. The publication remains in force until31 December
Glycan Mapping Dr C TYuen 2017 and the supplements are non-cumulative.
Heavy Metals Mr A Evans The date of entry into force of each publication is agreed by
Homoeopathic Manufacturing Methods Dr R A Pask-Hughes the members of the Convention. For new texts and
Mr J Sumal monographs the agreed date is the latest date by which all
Homoeopathic Raw Materials and Dr R A Pask-Hughes member sta tes must have implemented the standard but for
Stocks Mr J Sumal replacement texts and monographs the standard enters into
Host-cell proteins Dr A Kippen force on the same day in all states pany to the Convention.
lnhalanda Dr S Nichols The dates of entry into force of the monographs in the 8th
lno~ganic Chemistry Mr C T Goddard Edition and Supplements are given in the Table below.
Microbiological Qualiry of Herbal Drugs Dr K Helliwell
(Chairman) Publication Implementation Date
Modern Microbiological Methods Professor S Denyer 8th Edition 1 January 2014
Mr G Marco Supplement 8.1 1 April 2014
Monoclonal Antibodies Dr R Thorpe Supplement 8.2 1 July 2014
(Chairman) Supplement 8.3 1 January 2015
Dr P Varley Supplement 8.4 1 April 2015
Monocyte Activation Test Dr L Findlay Supplement 8.5 1 July 2015
Mycoplasmas Dr R Nicholas Supplement 8.6 1 January 2016
Near lnfrared Spectrometry Dr N Broad Supplement 8.7 1 April2016
Nuclear Magnetic Resonance Dr C Jones Supplement 8.8 1 July 2016
Pharmaceutical Preparations Mr V Fenton-May To provide the user of the British Pharmacopoeia with a
(Chairman) comprehensive reference 10 pharmacopoeial standards
Dr M GLee applicable in the United Kingdom, monographs of the
Powder Characterisation Techniques Dr G Webber European Pharmacopoeia, as amended by the Supplements,
P4 Procedure 4 for Biologicals Dr K Chidwick are inc1uded in the British Pharmacopoeia or the British
Precursors for Radiopharmaceutical Mr J Brain Pharmacopoeia (Veterinary), as appropriate (see the General
Preparations Notice on the European Pharmacopoeia).
Process Analytical Technology Dr N Broad Where the title of the monograph entry inc1uded in the
Mr I Lynch British Pharmacopoeia is different from the English title of
Production and Compounding of Mr P Maltby the European Pharmacopoeia monograph, an approved
Radiopharmaceutical Preparations synonyrn has been created by the British Pharmacopoeia
Propellants Dr S Nichols Commission (see Appendix XXI B).
Raw Materials for the production of Dr L Bisset
Cellular and Gene Therapy Products
Rules of Procedure Dr M G Lee C. Certification Scheme
Special Revision Programme Dr M GLee 1. The scheme is operated in accordance with the procedures
Standard Terms Dr M Ahmed described in Council of Europe Resolution AP-CSP(07) 1.
Statistics Dr R Gaines Das Additional information, inc1uding a list of certifica tes granted,
Traditional Chinese Medicines Mr M Whaley is published regularly in Pharmeuropa.
Water for Pharmaceutical Use DrM G Lee
(Chairman) 2. The scheme has been extended by means of Resolution
Mr A Hopkins AP-CSP(07) 1 and Directives 2001/83/EC and 2001/82/EC
Water for the Preparation of Extracts Dr K Helliwell
1 The Convenrion on ¡he Elabormion of a European Phannacopoeia
(European TreaIY Series No. 50; UK TreaIY Series No. 32 (1974) CMND
5763) as amended by ¡he ProLOcol (European TreaIY Series No. 134;
UK Tremy Series No. MISC16 (1990) CMND 1133).
V-A658 Supplementary Chapter IV D 2014
as amended, of the European Union ro cover active solvents which may remain in active substances, excipients
substances or excipients (organic or inorganic, obtained by and medicinal products after processing. This guideline, the
synthesis, extraction or fermentation), any product with text of which is reproduced below, exc1udes existing
transmissible spongiform encephalopathy (TSE) risk, or marketed products. The European Pharmacopoeia is,
herbal products used in the production or preparation of however, applying the same principIes enshrined in the
pharmaceutical products. guideline ro existing active substances, excipients and
3. For the established prOcess, the certificate is a 'Certificate medicinal products whether or not they are the subject of a
of Suitability of a Monograph of the European Pharmacopoeia' . monograph of the Pharrnacopoeia. AlI substances and
It certifies that the relevant Ph. Eur. monograph adequately products are to be tested for the content of solvents likely ro
controls the substance as manufactured by the company be present in a substance or producto
con cerned at the time that the certificate is granted (mainly Where the limits to be applied comply with those given
that the impurity tests are adequate ro control the below, tests for residual solvents are not generally mentioned
impurity profile associated with the particular source/route of in specific monographs since the solvents employed may vary
synthesis). It is not a certificate of compliance. Ir does not from one manufacturer to another and the requirements of
certify that the su)Jstance, as manufactured by the company this general chapter are applied via the general monograph
concerned at the time that the certificate is granted, complies on Substances for Pharmaceutical Use (2034) . The competent
with the requirements of the relevant Ph. Eur. monograph. authority is to be informed of the solvents employed during
4. The Certification Scheme is recognised by all signarory the production process. This information is also given in the
sta tes of the European Pharmacopoeia Convention and by dossier submitted for a certificate of suitability of the
the European Union. Canada, Australia, New Zealand, monographs of the European Pharmacopoeia and is
Tunisia and Morocco also recognise the scheme. mentioned on the certificate.
5. The certificate is intended ro facilitate the licensing Where only Class 3 solvents are used, a test for loss on
process. It can be used to simplify the data required within drying may be applied or a specific determination of the
the Control of starting material s section of an application as solvent may be made. If for a Class 3 solvent a justified and
stated in the amended Annex ro European Community authorised limit higher than 0.5 per cent is applied, a specific
Directive 200 l/83/EC and the associated EC Guideline on determination of the solvent is required.
Requirements in relation to active substances. When Class 1 residual solvents or Class 2 residual solvents
The Certification Scheme is intended ro operate in an (or Class 3 residual solvents which exceed the 0.5 per cent)
analogous fashion ro that for European Drug Master Files are used, the methodology described in the general method
(EDMFs) for which it is the preferred alternative. Its general (2.4.24) is ro be applied wherever possible. Otherwise an
purpose is ro avoid duplication of work by marketing appropriate validated method is to be employed.
authorisation applicants in preparing dossiers and by the When a quantitative determination of a residual solvent is
authorities in the assessment process. The scheme also assists carried out, the result is taken into account for the
in eliminating differences in the interpretation of Ph. Eur. calculation of the content of the substance except where a
monographs by various competent authorities. The operation test for drying is carried out.
of the scheme provides the European Pharmacopoeia
Commission with a mechanism for updating monographs, for Impurities: Guidelines for Residual Solvents
example, to cover substances made by different (CPMP/ICHI283/95)
manufacturing methods. ' 1. INTRODUCTION
6, The scope of the procedure was extended by means of
2. SCOPE OF THE GUIDELlNE
Resolution AP-CSP(07) 1. It now covers substances produced
by fermentation as indirect gene products, which are 3. GENERAL PRINCIPLES
metabolites of microorganisms, irrespective of whether or not 3.1. CLASSIFICATION OF RESIDUAL SOLVENTS
the microorganisms have been modified by traditional BY RISK ASSESSMENT
procedures or r-DNA technology and products with risk of 3.2. METHODS FOR ESTABLISHING EXPOSURE
transmitting agents of animal spongiform encepralopathies LIMITS
(TSE). Substances which are direct gene products, such as 3.3. OPTIONS FOR DESCRIBlNG LIMITS OF
proteins, and substances obtained from human tissues, CLASS 2 SOLVENTS
vaccines and blood products and preparations remain 3.4. ANALYTICAL PROCEDURES
exc1uded from the Certification procedure. The final decision
on eligibility of an application for a certificate of suitability 3.5. REPORTING LEVELS OF RESIDUAL
for a material of animal origin is taken by the relevant board SOLVENTS
of the procedure if necessary. 4. LIMITS OF RESIDUAL SOLVENTS
4.1. SOLVENTS TO BE AVOIDED
4.2. SOLVENTS TO BE LIMITED
D. Residual Solvents 4.3. SOLVENTS WITH LOW TOXIC POTENTIAL
Guidelines concerning residual solvents have been published in the
4.4. SOLVENTS FOR WHICH NO ADEQUATE
TOXICOLOGICAL DATA WAS FOUND
European Pharmacopoeia (section 5.4) and are reproduced here.
GLOSSARY
Linúting Residual Solvent Leve1s in Active APPENDIX 1. LIST OF SOLVENTS lNCLUDED IN
Substances, Excipients and Medicinal Products THE GUIDELINE
The International Conference on Harmonisation of APPENDIX 2. ADDITIONAL BACKGROUND
Technical Requirements for Registration of Pharmaceuticals A2.1: ENVIRONMENTAL REGULATION OF
for Human Use (ICH) has adopted Impurities Guidelines for ORGANIC VOLATILE SOLVENTS
Residual Solvents which prescribes limits for the content of
2014 Supplementary Chapter IV D V-A659
A2.2: RESIDUAL SOLVENTS IN to ascertain whether the formulation process has reduced the
PHARMACEUTICALS relevant solvent level to within the acceptable amount.
APPENDIX 3. METHODS FOR ESTABLISHING Medicinal product should also be tested if a solvent is used
EXPOSURE LIMITS during its manufacture.
1. lntroduction This guideline do es not apply to potential new active
substances, excipients, or medicinal products used during the
The objective of this guideline is to recommend acceptable
clinical research stages of development, nor does it apply to
amounts of residual solvents in pharmaceuticals for the safety
existing marketed medicinal products.
of the patient. The guideline recommends the use of less
toxic solvents and describes levels considered to be The guideline applies to all dosage forms and routes of
toxicologically acceptable for some residual solvents. administration. Higher levels of residual solvents may be
acceptable in certain cases such as short term (30 days or
Residual solvents in pharmaceuticals are defined here as
less) or topical application. Justification for these levels
organic volatile chemicals that are used or produced in the
should be made on a case by case basis.
manufacture of active substances or excipients, or in the
preparation of medicinal products. The solvents are not See Appendix 2 for additional background information
completely removed by practical manufacturing techniques. related to residual solvents.
Appropriate selection of the solvent for the synthesis of active 3. General principies
substance may enhance the yield, or determine characteristics 3.1. CLASSIFICATION OF RESIDUAL SOLVENTS BY RISK
such as crystal form, purity, and solubility. Therefore, the ASSESSMENT
solvent may sometimes be a critical parameter in the The term "tolerable daily intake" (TDI) is used by the
synthetic process. This guideline do es not address solvents International Program on Chemical Safety (IPCS) to
deliberately used as excipients nor does it address solvates. describe exposure limits of toxic chemicals and "acceptable
However, the content of solvents in such products should be daily intake" (ADI) is used by the World Health
evaluated and justified. Organisation (WHO) and other national and international
Since there is no therapeutic benefit from residual solvents, health authorities and institutes. The new term "permitted
all residual solvents should be removed to the extent possible daily exposure" (PDE) is defined in the present guideline as
to meet product specifications, good manufacturing practices, a pharmaceutically acceptable intake of residual solvents to
or other quality-based requirements. Medicinal products avoid confusion of differing values for ADI's of the same
should contain no higher leve!s of residual solvents than can substance.
be supported by safety data. Sorne solvents that are known to Residual solvents assessed in this guideline are listed in
cause unacceptable toxicities (Class 1, Table 1) should be Appendix 1 by common names and structures. They were
avoided in the production of active substances, excipients, or evaluated for their possible risk to human health and placed
medicinal products unless their use can be strongly justified into one of three classes as follows:
in a risk-benefit assessment. Some solvents associated with Class 1 solvents: Solvents to be avoided
less severe toxicity (Class 2, Tab1e 2) should be limited in
K.nown human carcinogens, strongly suspected human
order to protect patients from potential adverse effects.
carcinogens, and environmental hazards .
Ideally, less toxic solvents (Class 3, Table 3) should be used
where practica!. The complete list of solvents included in this Class 2 solvents: Solvents to be limited
guideline is given in Appendix 1. Non-genotoxic animal carcinogens or possible causative
The lists are not exhaustive and other solvents can be used agents of other irreversible toxicity such as neurotoxicity or
and later added to the lists. Recommended limits of Class 1 teratogenicity.
and 2 solvents or classification of solvents may change as new Solvents suspected of other significant but reversible
safety data becomes available. Supporting·safety data in a toxicities .
marketing application for a new medicinal product containing Class 3 solvents: Solvents with low toxic potential
a new solvent may be based on concepts in this guideline or Solvents with low toxic potential to man; no health-based
the concept of qualification of impurities as expressed in the exposure limit is needed. Class 3 solvents have PDEs of
guideline for active substances (Q3A, Impurities in New 50 mg or more per day.
Active Substances) or medicinal products (Q3B, Impurities
in New Medicinal Products), or all three guidelines. 3.2. METHODS FOR ESTABLISHING EXPOSURE LIMITS
The method used to establish permitted daily exposures for
2. Scope 01 the guideline residual solvents is presented in Appendix 3. Summaries of
Residual solvents in active substances, excipients, and in the toxicity data that were used to establish limits are
medicinal products are within the scope of this guideline. published in Pharmeuropa, Vo!. 9, No. 1, Supplement April
Therefore, testing should be performed for residual solvents 1997.
when production or purification processes are known to
3.3. OPTIONS FOR DESCRIBING LIMITS OF CLASS 2
result in the presence of such solvents. It is only necessary to
SOLVENTS
test for solvents that are used or produced in the
manufacture or purification of active substances, excipients, Two options are available when setting limits for Class 2
or medicinal product. Although manufacturers may choose to solvents.
test the medicinal product, a cumulative method may be Option 1 The concentration limits in parts per million
used to calculate the residual solvent levels in the medicinal stated in Table 2 can be used. They were calculated using
product from the levels in the ingredients used to produce equation (1) below by assuming a product mass of 10 g
the medicinal product. If the calculation results in a leve! administered daily.
equal to or below that recommended in this guideline, no
testing of the medicinal product for residual solvents need be x PDE
considered. If however, the calculated level is aboye the Concentration (ppm ) = -1000
-:----
dos e
(1)
recommended level, the medicinal product should be tested
Here, PDE is given in terms of mglday and dose is given in In this example, the product meets neither the Option 1 nor
g/day. the Option 2 limit according to this summation.
These limits are considered acceptable for all substances, The manufacturer could test the medicinal product to
excipients, or products. Therefore this option may be applied determine if the formulation process reduced the level of
if the daily dose is not known or fixed. If all excipients and acetonitrile. If the level of acetonitrile was not reduced during
active substances in a formulation meet the limits given in formulation to the alIowed limit, then the manufacturer of
Option 1, then these components may be used in any the medicinal product should take other steps to reduce the
proportion. No further calculation is necessary provided the amount of acetonitrile in the medicinal producto If alI of
daily dose does not exceed 10 g. Products that are these steps fail to reduce the level of residual solvent, in
administered in doses greater than 10 g per day should be exceptional cases the manufacturer could provide a summary
considered under Option 2. of efforts made to reduce the solvent level to meet the
Opdon 2 It is not considered necessary for each guideline value, and provide a risk-benefit analysis to support
component of the medicinal product to comply with the allowing the product to be utilised containing residual solvent
limits given in Option 1. The PDE in terms of mglday as at a higher leve!.
stated in Table 2 can be used with the known maximum 3.4 . ANALYTICAL PROCEDURES
daily dose and equation (1) aboye to determine the Residual solvents are typically determined using
concen'tration of residual solvent allowed in a medicinal chromatographic techniques such as gas chromatography.
producto Such limits are considered acceptable provided that Any harmonised procedures for determining levels of residual
is has been demonstrated that the residual solvent has been solvents as described in the pharmacopoeias should be used,
reduced to the practical minimum. The limits should be if feasible. Otherwise, manufacturers would be free to select
realistic in relation to analytical precision, manufacturing the most appropriate validated analytical procedure for a
capability, reasonable variation in the manufacturing process, particular application. If only Class 3 solvents are present, a
and the limits should refiect contemporary manufacturing non-specific method such as loss on drying may be used.
standards. Validation of methods for residual solvents should conform
Option 2 may be applied by adding the amounts of a residual to ICH guidelines "Text on Validation of Analytical
solvent present in each of the components of the medicinal Procedures" and "Extension of the ICH Text on Validation
productoThe sum of the amounts of solvent per day should of Analytical Procedures".
be less than that given by the PDE. 3.5. REPORTING LEVELS OF RESIDUAL SOLVENTS
Consider an example of the use of Option l and Option 2 Manufacturers of pharmaceutical products need certain
applied to acetonitrile in a medicinal producto The permitted information about the content of residual solvents in
daily exposure to acetonitrile is 4.1 mg per day; thus, the excipients or active substances in order to meet the criteria of
Option 1 limit is 410 ppm. The maximum administered daily this guideline. The following statements are given as
mass of a medicinal product is 5.0 g, and the medicinal acceptable examples of the information that could be
product contains two excipients. The composition of the provided from a supplier of excipients or active substances to
medicinal product and the calculated maximum content of a pharmaceutical manufacturer. The supplier might choose
residual acetonitrile are given in the following tableo one of the following as appropriate:
- Only Class 3 solvents are likely to be presento Loss on
Component Amount in Acetonitrile Daily drying is les s than 0.5 per cent.
formulation content exposure - Only Class 2 solvents X, Y, ... are likely to be presento
Active substance 0.3 g 800 ppm 0.24 mg
AII are below the Option 1 limit
Excipient 1 0.9 g 400 ppm 0.36 mg - (Here the supplier would name the Class 2 solvents
Excipient 2 3.8 g 800 ppm 3.04 mg represented by X, Y, ... )
Medicinal product 5.0 g 728 ppm 3.64 mg - Only Class 2 solvents X, Y, ... and Class 3 solvents are
likely to be presento Residual Class 2 solvents are below
the Option 1 limit and residual Class 3 solvents are below
Excipient l meets the Option l limit, but the drug substance, 0.5 per cent.
excipient 2, and medicinal product do not meet the Option l If Class 1 solvents are likely to be present, they should be
limit. Nevertheless, the product meets the Option 2 limit of identified and quantified. "Likely to be present" refers to the
4.1 mg per day and thus conforms to the recommendations in solvent used in the final manufacturing step and to solvents
this guideline. that are used in earlier manufacturing steps and not removed
Consider another example using acetonitrile as residual consistently by a validated process.
solvent. The maximum administered daily mas s of a If solvents of Class 2 or Class 3 are present at greater than
medicinal product is 5.0 g, and the medicinal product their Option 1 limits or 0.5 per cent, respectively, they
contains two excipients. The composition of the medicinal should be identified and quantified.
product and the calculated maximum content of residual
acetonitrile is given in the following tableo 4. Limits 01 residual solvents
4.1. SOLVENTS TO BE AVOIDED
Solvents in Class 1 should not be employed in the
Component Amount in Acetonitrile Daily
formulation
manufacture of active substances, excipients, and medicinal
conten! exposure
Active substance 0.3 g 800 ppm 0.24 mg
products because of their unacceptable toxicity or their
deleterious environmental effect. However, if their use is
Excipient 1 0.9 g 2000 ppm 1.80 mg unavoidable in order to produce a medicinal product with a
Excipient 2 3.8 g 800 ppm 3.04 mg significant therapeutic advance, then their levels should be
Medicinal product 5.0 g 1016 ppm 5.08 mg
restricted as shown in Table 1, unless otherwise justified.
1,l,l-Trichloroethane is included in Table 1 because it is an
environmental hazard. The stated limit of 1500 ppm is based 4.3. SOLVENTS WITH LOW TOXIC POTENTIAL
on a review of the safety data. Solvents in Class 3 (shown in Table 3) may be regarded as
less toxic and of lower risk to human health. Class 3 inc1udes
Table 1. - Class 1 solvents in pharmaceutical products no solvent known as a human health hazard at levels
(solvents that should be avoided) normally accepted in pharmaceuticals. However, there are no
Solvent Concentration Iimit Concero long-term toxicity or carcinogenicity studies for many of the
(1!I!m) solvents in Class 3. Available data indicate that they are less
Benzene 2 Carcinogen
toxic in acute or short-term studies and negative in
Carbon tetrachloride 4 Toxic and environmental hazard genotoxicity studies. It is considered that amounts of these
1.2-Dichloroethane 5 Toxic residual solvents of 50 mg per day or les s (corresponding to
5000 ppm or 0.5 per cent under Option 1) would be
1.1-Dichloroethene 8 Toxic
acceptable without justification. Higher amounts may also be
1.1.1·Trichloroethane 1500 Environmental hazard acceptable provided they are realistic in relation to
manufacturing capability and good manufacturing practice.
4.2. SOLVENTS TO BE LIMITED Table 3. - Class 3 solvents which should be limited by CMP or
Solvents in Table 2 should be limited in pharmaceutical other quality-based requirements
products because of their inherent toxicity. PDEs are given to Acetic acid Heptane
the nearest 0.1 mglday, and concentrations are given to the Acetone Isobutyl acetate
nearest 10 ppm. The stated values do not refiect the
Anisole Isopropyl acetate
necessary analytical precision of determination. Precision
should be determined as part of the validation of the method. l-Butanol Methyl acetate
2-Butanol 3-Methyl-1-butanol
Table 2. - Class 2 solvents in pharmaceutical products
Butyl acetate Methylethylketone
Solvent PDE Concentration IImit
(mg(day) (1!I!m) terl-Butylmethyl ether Methylisobutylketone
Acetonitrile 4.1 410
Cumene 2-Methyl-I-propanol
Chlorobenzene 3.6 360
Dimethyl sulfoxide Pentane
Chloroform 0.6 60
Ethanol l-Pentanol
Cyclohexane 38.8 3880
Ethyl acetate 1-Propanol
1.2-Dichloroethene 18.7 1870
Ethyl ether 2-Propanol
Dichloromethane 6.0 600
Ethyl formate Propyl acetate
1.2-Dimethoxyethane 1.0 100
Formic acid
N,N-Dimethylacetamide 10.9 1090
N,N-Dimethylformamide 8.8 880
4.4. SOLVENTS FOR WHICH NO ADEQUATE TOXICOLOGICAL
1.4-Dioxane 3.8 380
DATA WAS FOUND
2-Ethoxyethanol 1.6 160 The following solvents (Table 4) may also be of interest to
Ethyleneglycol 6.2 620 manufacturers of excipients, active substances, or medicinal
products. However, no adequate toxicological data on which
Formamide 2.2 220
to base a PDE was found. Manufacturers should supply
Hexane 2.9 290 justification for residual levels of these solvents in
Methanol 30.0 3000 pharmaceutical products .
2-Methoxyethanol 0.5 50
Table 4. - Solvents for which no adequate toxicological data
Methylbutylketone 0.5 50 was found
Methylcyclohexane 11.8 1180 1.l-Diethoxypropane Methylisopropylketone
N-Methylpyrrolidone 5.3 530 l .l-Dimethoxymethane Methyltetrahydrofuran
Nitromethane 0.5 50 2.2-Dimethoxypropane Petroleum ether
Pyridine 2.0 200 Isooctane Trichloroacetic acid
Sulfolane 1.6 160 Isopropyl ether Trifluoroacetic acid
Tetrahydrofuran 7.2 720
Tetralin 1.0 100
Glossary
Toluene 8.9 890
Genotoxic carcinogens Carcinogens which produce
1,1.2-Trichloroethene 0.8 80 cancer by affecting genes or chromosomes.
Xylene* 21.7 2170 LOEL Abbreviation for lowest-observed effect leve!.
'usually 60 per cent m-xylene. 14 per cent p-xylene. 9 per cent o-xylene Lowest-observed effect level The lowest dose of
with 17 per cent ethyl benzene substance in a study or group of studies that produces
biologically significant increases in frequency or severity of
any effects in the exposed humans or animals.
Modi.fying factor A factor dete=ined by professional

judgement of a toxicologist and applied to bioassay data to
relate that data safely to humans.
Neurotoxicity The ability of a substance to cause adverse
effects on the nervous system.
NOEL Abbreviation for no-observed-effect level.
No-observed-effect level The highest dose of substance
at which there are no biologically significant increases in
frequency or severity of any effects in the exposed humans or
animals.
PDE Abbreviation for pennitted daily exposure.
Permitted daily exposure The maximum acceptable
intake per day of residual solvent in pha=aceutical
products.
Reversible toxicity The occurrence of ha=ful effects that
are caused by a substance and which disappear after
exposure to the substance ends.
Strongly suspected human carcinogen A substance for
which there is no epidetniological evidence of carcinogenesis
but there are positive genotoxicity data and clear evidence of
carcinogenesis in rodents.
Teratogenicity The occurrence of structural
malfo=ations in a developing foetus when a substance is
administered during pregnancy.
APPENDIX 1. LIST OF SOLVENTS INCLUDED IN THE GUIDELINE

Solvent Other Names Structure Class
Acetic acid Ethanoic acid CH3COOH Class 3
Acetone 2-Propanone CH3COCH 3 Class 3
Propan-2-one
Acetonitrile CH 3CN Class 2
Anisole Methoxybenzene Class 3
Benzene
l-Butanol
Benzol
n-Butyl alcohol
Butan-l-01
o
CH3[CH,J30H
Class 1
Class 3
2-Butanol sec-Butyl alcohol CH3CH,CH(OH)CH3 Class 3

Butan-2-o1
Butyl acetate Acetic acid butyl ester CH3COO[ CH,J3CH3 Class 3
tert-Butylmethyl ether 2-Methoxy-2-methylpropane (CH3)3COCH3 Class 3
Carbon tetrachloride Tetrachloromethane CCI, Class 1
Chlorobenzene Class 2
(YCI
V CHCl 3
Chloroform Trichloromethane Class 2
Cumene lsopropylbenzene Class 3
(l-Methylethyl)benzene
Cyclohexane
1,2-Dichloroethane
Hexamethylene
sym-Dichloroethane
Ethylene dichloride
o
CH,CICH,CI
Class 2
Class 1
Ethylene chloride
1,1-Dichloroethene 1,I-Dichloroethylene H,C=CCI, Class 1
Vinylidene chloride
1,2-Dichloroethene 1,2-Dichloroethylene CIHC=CHCI Class 2
Acetylene dichloride
Dichloromethane Methylene chloride CH,CI, Class 2
1,2-Dimethoxyethane Ethyleneglycol dimethyl ether H3COCH,CH,OCH3 Class 2
Monoglyme
Dimethyl cellosolve
N,N-Dimethylacetamide DMA CH 3CON(CH3), Class 2
N,N-Dimethylformamide DMF HCON(CH 3), Class 2
Dimethyl sulfoxide Methylsulfinylmethane (CH3),SO Class 3
Methyl sulfoxide
DMSO
1,4-Dioxane p-Dioxane Class 2
[l,4JDioxane (0)
Ethanol Ethyl alcohol °
CH 3CH,OH Class 3
2-Ethoxyethanol Cellosolve CH 3CH,OCH,CH,OH Class 2
Ethyl acetate Acetic acid ethyl ester CH3COOCH;CH 3 Class 3
Ethyleneglycol 1,2-Dihydroxyethane HOCH,CH,OH Class 2
1,2-Ethanediol
Ethyl ether Diethyl ether CH3CH,OCH,CH 3 Class 3
Ethoxyethane
1, l'-Oxybisethane
Ethyl formate Formic acid ethyl ester HCOOCH,CH 3 Class 3
Formamide Methanamide HCONH, Class 2
Formic acid HCOOH Class 3
Solvent OtherNames Structure Class

Heptane n-Heptane CH,[CH,I,CH, Class 3
Hexane n-Hexane CH,[CH,I,CH3 Class 2
Isobutyl acetate Acetic acid isobutyl ester CH3COOCH,CH(CH3), Class 3
Isopropyl acetate Acetic acid isopropyl ester CH3COOCH(CH3 ), Class 3
Methanol Methyl alcohol CH30H Class 2
2-Methoxyethanol Methyl cellosolve CH 3OCH,CH,OH Class 2
Methyl acetate Acetic acid rnethyl ester CH3COOCH3 Class 3
3-Methyl-l-butanol Isoarnyl alcohol (CH3),CHCH,CH,OH Class 3
Isopentyl alcohol
3-Methylbutan-l-01
Methylbutylketone 2-Hexanone CH 3[ CH,],COCH3 Class 2
Hexan-2-one
Methylcyclohexane Cyclohexylrnethane CH
Class 2
() 3
Methylethylketone 2-Butanone CH 3CH,COCH3 Class 3

MEK
Butan-2-one
Methylisobutylketone 4-Methylpentan-2-one CH3COCH,CH(CH3), Class 3
4-Methyl-2-pentanone
MIBK
2-Methyl-l-propanol Isobutyl alcohol (CH 3),CHCH,OH Class 3
2-Methylpropan-l-01
N-Methylpyrrolidone l-Methylpyrrolidin-2-one CH 3 Class 2
l-Methyl-2-pyrrolidinone I
(yo
Nitrornethane CH 3NO, Class 2
Pentane n-Pentane CH 3[CH,],CH3 Class 3
l-Pentanol Arnyl alcohol CH3[CH,13CH,OH Class 3
Pentan-l-01
Pentyl alcohol
l-Propanol Propan-l-ol CH 3CH2CH,OH Class 3
Propyl alcohol
2-Propanol Propan-2-o1 (CH3),CHOH Class 3
Isopropyl alcohol
Propyl acetate Acetic acid propyl ester CH3 COOCH,CH,CH, Class 3
pyridine Class 2
N
-Sulfonane Tetrahydrothiophene l,l-dioxide

O Class 2
°°~/,I
Ó
Tetrahydrofuran Tetrarnethylene oxide Class 2
Oxacyclopentane
O
ca
Tetralin 1,2,3,4-Tetrahydronaphthalene Class 2
Toluene Methylbenzene
O
1#
CH
3
Class 2
1,1,I-Trichloroethane Methylchloroforrn CH,CCI, Class 1

1,1,2-Trichloroethene Trichloroethene HCIC=CCI, Class 2
Xylene' Dirnethybenzene
Xylol
H3C~
a 1
'usually 60 per cent m-xylene, 14 per cent p-xylene, 9 per cent o-xylene with 17 per cent ethyl benzene.
CH3 Class 2
Appendix 2. Additional background the method adopted by IPCS for Assessing Human Health
A2.1. ENVIROMENTAL REGULATION OF ORGANIC VOLATILE Risk of Chemicals (Enviromnental H ealth Cn'teria 170, WHO,
SOLVENTS 1994). These methods are similar to those used by the
Several of the residual solvents frequently used in the USEPA (IRIS) and the USFDA (R ed Book) and others.
production of pharmaceuticals are listed as toxic chemicals in The method is outlined here to give a better understanding
Environmental Health Criteria (EHC) monographs and the of the origin of the PDE values. It is not necessary to
Integrated Risk Information System (IRIS). The objectives of perform these ca1culations in order to use the PDE values
such groups as the Intemational Programme on Chemical tabulated in Section 4 of this documento
Safety (IPCS), the United Sta tes Environmental Protection PDE is derived from the no-observed-effect level (NOEL) , or
Agency (USEPA) and the United States Food and Drug the lowest-observed effect level (LOEL), in the most relevant
Administration (USFDA) inc1ude the determination of animal study as follows:
acceptable exposure levels. The goal is protection of human
health and maintenance of environmental integrity against PDE = NO EL x Weight Adjustment
the possible deleterious effects of chemicals resulting from Fl x F2 x F3 x F4 x F5
long-term environmental exposure. The methods involved in
the estimation of maximum safe exposure Iimits are usually
based on long-term studies. When long-term study data are The PDE is derived preferably from a NO EL. If no NOEL is
unavailable, shorter term study data can be used with obtained, the LOEL may be used. Modifying factors
modification of the approach such as use of larger safety proposed here, for relating the data to humans, are the same
factors. The approach described therein relates primarily to kind of "uncertainty factors " used in Environmental Health
long-term or Iife-time exposure of the general population in Criteria (Enviromnental H ealth C1iteria 170, World Health
the ambient environment, i.e. ambient air, food, drinking Organisation, Geneva, 1994), and " modifying factors" or
water and other media. "safety factors" in Pharmacopoeial Forwn. The assumption of
100 per cent systemic exposure is used in all ca1culations
A2.2. RESIDUAL SOLVENTS IN PHARMACEUTICALS
regardless of route of administration.
Exposure Iimits in this guideline are established by referring
to methodologies and toxicity data described in EHC and The modifying factors are as follows:
IRIS monographs . However, sorne specific assumptions Fl = A factor to account for extrapolation between species
about residual solvents to be used in the synthesis and F 1 = 2 for extrapolation from dogs to humans
formulation of pharmaceutical products should be taken into Fl = 2.5 for extrapolation from rabbits to humans
account in establishing exposure limits. They are: Fl = 3 for extrapolation from monkeys to humans
1) Patients (not the general population) use Fl = 5 for extrapolation from rats to humans
pharmaceuticals to treat their diseases or for prophylaxis F 1 = 10 for extrapolation from other animals to humans
to prevent infection or disease. F 1 = 12 for extrapolation from mice to humans
2) The assumption of life-time patient exposure is not Fl takes into account the comparative surface area: body
necessary for most pharmaceutical products but may be weight ratios for the species concemed and for mano Surface
appropriate as a working hypothesis to reduce risk to area (S) is ca1culated as:
human health.
3) Residual solvents are unavoidable components in s = kmO. 67
pharmaceutical production and will often be a part of
medicinal products. in which 111 = body mass, and the constant k has been taken
4) Residual solvents should not exceed recommended levels to be 10. The body weight used in the equation are those
except in exceptional circumstances. shown below in Table A3.-1.
5) Data from toxicological studies rhat are used to F2 = A factor of 10 to account for variability between
determine acceptable levels for residual solvents should individuals. A factor of 10 is generally given for all
have been generated using appropriate protocols such as organic solvents, and 10 is useq consistently in this
those described for example, by OECD and the FDA guideline.
Red Book. F3 = A variable factor to account for toxicity studies of
Appendix 3. Methods lor establishing exposure limits short-term exposure
F3 = 1 for studies that last at least one half-lifetime (1 year
The Gaylor-Kodell method of risk assessment (Gaylor, D.
for rodents or rabbits; 7 years for cats, dogs and
W. and Kodell, R. L. Linear Interpolation algorithm for low
monkeys).
dose assessment of toxic substance. J. Environ. Pathology, 4,
F3 = 1 for reproductive studies in which the whole period of
305, 1980) is appropriate for Class 1 carcinogenic solvents .
organogenesis is covered.
Only in cases where reliable carcinogenicity data are available
F3 = 2 for a 6 month study in rodents, or a 3.5 year study
should extrapolation by the use of mathematical models be
in non-rodents.
applied to setting exposure Iimits. Exposure Iimits for Class 1
F3 = 5 for a 3 month study in rodents, or a 2 year study in
solvents could be determined with the use of a large safety
non-rodents.
factor (i.e., 10 000 to 100 000) with respect to the
F3 = 10 for studies of a shorter duration.
no-observed-effect level (NOEL). Detection and
quantification of these solvents should be by state-of-the-art In all cases, the higher factor has been used for study
analytical techniques. durations between the time points, e.g. a factor of 2 for a
Acceptable exposure levels in this guideline for Class 2 9 month rodent study.
solvents were established by ca1culation of PDE values
according to the procedures for setting exposure Iimits in
pharmaceuticals (Phannacopeial ForUln, Nov-Dec 1989), and
Table A3.-1. - Values used in the calculations in this document n P 300 X 10- 6 atm x 153840 mg mol - 1
Rat body weight 425 g V RT 0.082 L atm K - 1mol 1 x 298 K
Pregnant rat body weight 330 g
46,15 mg = 1.89 m / L
Mouse body weight 28 g 24.45 L g
Pregnant mouse body weight 30 g
The relationship 1000 L = 1 m 3 is used to convert to
Guinea-pig body weight 500 g mg/m 3 .
Rhesus monkey body weight 2.5 kg
Rabbit body weight (pregnant or not) 4 kg
Beagle dog body weight 11.5 kg
Rat respiratory volume 290 Ljday
Mouse respiratory volume 43 Ljday
Rabbit respiratory volume 1440 Lj day
Guinea-pig respiratory volume 430 Ljday
Human respiratory volume 28800 Ljday
Dog respiratory volume 9000 Lj day
Monkey respiratory volume liSO Lj day
Mouse water consumption 5 mLj day
Rat water consumption 30 mLj day
Rat [ood consumption 30 gjday
When only a LOEL is available, a factor of up to 10 can be

used depending on the severity of the toxicity.
The weight adjustment assumes an arbitrary adult human
body weight for either sex of 50 kg. This relatively low
weight provides an additional safety factor against the
standard weights of 60 kg or 70 kg that are often used in this
type of calculation. It isrecognised that sorne adult patients
weigh less than 50 kg; these patients are considered to be
accommodated by the built-in safety factors used to
determine a PDE. If the solvent was present in a formulation
specifically intended feir paediatric use, an adjustment for a
lower body weight would be appropriate.
As an example of the application of this equation, consider
the toxicity study of acetonitrile in mice that is summarised
in Pharmeuropa, Vol. 9. No. 1, Supplement, Apri11997,
page S24. The NO EL is calculated to be 50.7 mg kg- I day-l.
The PDE for acetonitrile in this study is 'calculated as
follows:
PDE = 50.7mg kg- 1 day - l x 50 kg = 4.22 mg day-l

12 x 10 x 5 x 1 x 1
In this example,
Fl = 12 to account for the extrapolation ftom mice to
humans
F2 = lOto account for differences between individual
humans
F3 = 5 because the duration of the study was only 13 weeks
F4 = 1 because no severe toxicity was encountered
F5 = 1 because the no-effect level was determined
, The equation for an ideal gas, PV = nRT, is used to convert
concentrations of gases used in inhalation studies from units
of ppm to units of mg!L or mg/m 3 . Consider as an example
the rat reproductive toxicity study by inhalation of carbon
tetrach10ride (molecular weight 153.84) summarised in
Phanneuropa, Vol. 9, No. 1, Supplement, April 1997, page
S9.
2014 Supplementary Chapter IV E V-A667
E. Alcoholimetric Tables
The general formula agreed by the Council of the European Communities in its Directive of 27 July 1976 on alcoholimetry served as the
basis for establishing the following tables.
3 3
% VIV %m/m P20 (kg/m
3
) % VIV %m/m P20 (kg7m ) % VIV %m/m P20 (kg/m )
0.0 0.0 998.20 5.0 3.98 991.06 10.0 8.01 984.71

0.1 0.08 998.05 5.1 4.06 990.92 10.1 8.10 984.59
0.2 0.16 997.90 5.2 4.14 990.79 10.2 8.18 984.47
0.3 0.24 997.75 5.3 4.22 990.65 10.3 8.26 984.35
0.4 0.32 997.59 5.4 4.30 990.52 10.4 8.34 984.23
0.5 0.40 997 .44 5.5 4.38 990 .39 10.5 8.42 984.11
0.6 0.47 997.29 5.6 4.46 990.26 10.6 8.50 983.99
0.7 0.55 997.14 5.7 4.54 990.12 10.7 8.58 983.88
0.8 0.63 996.99 5.8 4.62 989.99 10.8 8.66 983.76
0.9 0.71 996.85 5.9 4.70 989.86 10.9 8.75 983.64
1.0 0.79 996.70 6.0 4.78 989.73 11.0 8.83 983.52

1.1 0.87 996.55 6.1 4.86 989.60 11.1 8.91 983.40
1.2 0.95 996.40 6.2 4.95 989.47 11.2 8.99 983.29
1.3 1.03 996 .25 6.3 5.03 989.34 11.3 9.07 983.17
1.4 1.11 996.11 6.4 5.11 989 .2 1 11.4 9.15 983.05
1.5 1.19 995.96 6.5 5.19 989.08 11.5 9.23 982.94
1.6 1.27 995.81 6.6 5.27 988.95 11.6 9.32 982.82
1.7 1.35 995.67 6.7 5.3 5 988.82 11.7 9.40 982.70
1.8 1.43 995.52 6.8 5.43 988.69 11.8 9.48 982.59
1.9 1.51 995.38 6.9 5.51 988.56 11.9 9.56 982.47
2.0 1.59 995.23 7.0 5.59 988.43 12.0 9.64 982.35

2.1 1.67 995.09 7.1 5.67 988.30 12.1 9.72 982.24
2.2 1.75 994.94 7.2 5.75 988.18 12.2 9.80 982.12
2.3 1.82 994.80 7.3 5.83 988.05 12.3 9.89 982.01
2.4 1.90 994.66 7.4 5.91 987.92 12.4 9.97 981.89
2.5 1.98 994.51 7.5 5.99 987.79 12.5 10.05 981.78
2.6 2.06 994.37 7.6 6.07 987.67 12.6 10.13 981.67
2.7 2.14 994.23 7.7 6.15 987.54 12.7 10.21 981.55
2.8 2.22 994.09 7.8 6.23 987.42 12.8 10.29 981.44
2.9 2.30 993 .95 7.9 6.32 987 .29 12.9 10.37 981.32
3.0 2.38 993.81 8.0 6.40 987.16 13.0 10.46 981.21

3.1 2.46 993.66 8.1 6.48 987.04 13.1 10.54 981.10
3.2 2.54 993.52 8.2 6.56 986.91 13.2 10.62 980.98
3.3 2.62 993 .38 8.3 6.64 986.79 13.3 10.70 980.87
3.4 2.70 993 .24 8.4 6.72 986.66 13.4 10.78 980 .76
3.5 2.78 993.11 8.5 6.80 986.54 13.5 10.87 980.64
3.6 2.86 992.97 8.6 6.88 986.42 13.6 10.95 980.53
3.7 2.94 992.83 8.7 6.96 986.29 13.7 11.03 980.42
3.8 3.02 992.69 8.8 7.04 986.17 13.8 1l.l1 980 .3 1
3.9 3.10 992.55 8.9 7.12 986.05 13.9 11.19 980.19
4.0 3.18 992.41 9.0 7.20 985.92 14.0 11.27 980.08

4.1 3.26 992.28 9.1 7.29 985.80 14. 1 11.36 979.97
4.2 3.34 992.14 9.2 7.37 985.68 14.2 11.44 979.86
4.3 3.42 992.00 9.3 7.45 985.56 14.3 11.52 979.75
4.4 3.50 991.87 9.4 7.53 985.44 14.4 11.60 979.64
4.5 3.58 991.73 9.5 7.61 985.31 14.5 11.68 979.52
4.6 3.66 991.59 9.6 7.69 985.19 14.6 11.77 979.41
4.7 3.74 991.46 9.7 7.77 985.07 14.7 11.85 979.30
4.8 3.82 991.32 9.8 7.85 984 .95 14.8 11.93 979.19
4.9 3.90 991.19 9.9 7.93 984.83 14.9 12.01 979.08
V -A668 Supplementary Chapter IV E 2014
% VIV % m/m P20 (kg/m3 ) % VIV % m/m P20 (kg/m

3
) % VIV % m/m P20 (kg/m 3 )
15.0 12.09 978 .9 7 20.0 16.21 973 .56 25.0 20.38 968 .10
15.1 12.17 978.86 20.1 16.30 973.45 25.1 20.47 967 .99
15.2 12.26 978.75 20.2 16.38 973.34 25.2 20.55 967 .87
15.3 12.34 978.64 20.3 16.46 973 .24 25.3 20.63 967 .76
15.4 12.42 978.53 20.4 16.55 973.13 25.4 20.72 967.65
15.5 12.50 978 .42 20.5 16.63 973.02 25.5 20.80 967.53
15.6 12.59 978.31 20.6 16.71 972 .91 25.6 20.88 967.42
15.7 12.67 978.20 20.7 16.79 972 .80 25.7 20.97 967.31
15.8 12.75 978 .09 20.8 16.88 972.70 25.8 21.05 967 .19
15.9 12.83 977.98 20.9 16.96 972 .59 25.9 21.14 967 .08
16.0 12.91 977.87 21.0 17.04 972 .48 26.0 21.22 966.97
16.1 13.00 977.76 21.1 17.13 972.37 26.1 21.31 966 .85
16.2 13.08 977.65 21.2 17.21 972.27 26.2 21.39 966.74
16.3 13.16 977.55 21.3 17.29 972.16 26.3 21.47 966.62
16.4 13.24 977.44 21.4 17.38 972.05 26.4 21.56 966.51
16.5 13.32 977.33 21.5 17.46 971.94 26.5 21.64 966.39
16.6 13.41 977.22 21.6 17.54 971.83 26.6 21.73 966 .28
16.7 13.49 977.11 21.7 17.62 971.73 26 .7 21.81 966 .16
16.8 13.57 977 .00 21.8 17.71 971.62 26.8 21.90 966.05
16.9 13.65 976.89 21.9 17.79 971.51 26.9 21.98 965.93
17.0 13.75 976.79 22.0 17.87 971.40 27.0 22.06 965.81

17. 1 13.82 976 .68 22. 1 17.96 971.29 27. 1 22.15 965.70
17.2 13.90 976.57 22.2 18.04 971.18 27.2 22 .23 965.58
17.3 13.98 976.46 22.3 18.12 971.08 27.3 22 .32 965.46
17.4 14.07 976.35 22.4 18.21 970 .97 27.4 22.40 965.35
17.5 14.15 976.25 22.5 18.29 970.86 27.5 22.49 965.23
17.6 14.23 976.14 22.6 18.37 970.75 27.6 22.57 965.11
17.7 14.3 1 976 .03 22.7 18.46 970 .64 27.7 22 .65 964.99
17.8 14.40 975.92 22.8 18.54 970.53 27.8 22.74 964.88
17.9 14.48 975.81 22.9 18.62 970.42 27.9 22.82 964.76
18.0 14.56 975.71 23.0 18.71 970.31 28.0 22.91 964.64

18.1 14.64 975.60 23.1 18.79 970.20 28.1 22.99 964.52
18.2 14.73 975.49 23.2 18.87 970.09 28.2 23.08 964.40
18.3 14.81 975.38 23.3 18.96 969.98 28.3 23.16 964.28
18.4 14.89 975.28 23.4 19.04 969.87 28.4 23.25 964.16
18.5 14.97 975.17 23 .5 19.13 969 .76 28.5 23.33 964.04
18.6 15.06 975.06 23.6 19.21 969.65 28.6 23.42 963.92
18.7 15.14 974.95 23.7 19.29 969.54 28.7 23.50 963.80
18.8 15.22 974.85 23 .8 19.38 969.43 28.8 23.59 969.68
18.9 15.30 974.74 23 .9 19.46 969.32 28.9 23.67 963.56
19.0 15.39 974.63 24.0 19.54 969 .21 29.0 23.76 963.44
19.1 15.47 974.52 24.1 19.63 969.10 29.1 23.84 963 .32
19.2 15.55 974.42 24.2 19.71 968.99 29.2 23.93 963.20
19.3 15.63 974.31 24. 3 19.79 968.88 29.3 24 .01 963 .07
19.4 15.72 974.20 24.4 19.88 968.77 29.4 24. 10 962.95
19.5 15.80 974.09 24.5 19.96 968.66 29.5 24.18 962.83
19.6 15.88 973.99 24.6 20.05 968.55 29 .6 24.27 962 .71
19.7 15.97 973 .88 24.7 20.13 968.43 29.7 24.35 962.58
19.8 16.05 973.77 24.8 20.21 968 .32 29.8 24.44 962.46
19.9 16.13 973.66 24.9 20.30 968.21 29.9 24.52 962.33
2014 Supplementary Chapter IV E V -A669
% VIV %mlm Pzo (kg/m 3 ) % VIV % mlm Pzo (kg/m 3 ) % VIV % mlm Pzo (kg/m 3 )
30.0 24.6 1 962 .21 35.0 28 .91 955.59 40.0 33.30 948 .05
30.1 24.69 962.09 35. 1 28.99 955.45 40.1 33.39 947 .88
30.2 24.78 961.96 35.2 29.08 955 .30 40.2 33.48 947.72
30.3 24.86 961.84 35.3 29.17 955 .16 40.3 33.57 947.56
30.4 24.95 961.71 35.4 29.26 955 .02 40.4 33.66 947.40
30.5 25 .03 961.59 35.5 29.34 954.88 40. 5 33.74 947.24
30.6 25.12 961.46 35.6 29.43 954.73 40.6 33.83 947.08
30.7 25.20 961.33 35.7 29.52 954.59 40.7 33.92 946.91
30.8 25.29 961.21 35.8 29.60 954.44 40.8 34.01 946.75
30.9 25.38 961.08 35.9 29.69 954.30 40.9 34.10 946.58
31.0 25.46 960.95 36.0 29.78 954.15 4 1.0 34.19 946 .42
31.1 25.55 960 .82 36.1 29.87 954.01 41.1 34.28 946.26
31.2 25.63 960 .70 36.2 29.95 953.86 41.2 34.37 946.09
31.3 25 .72 960.57 36.3 30.04 953.72 41.3 34.46 945.93
31.4 25.80 960.44 36.4 30.13 953.57 41.4 34.55 945.76
31.5 25.89 960.31 36.5 30.21 953.42 41.5 34.64 945.59
31.6 25.97 960.18 36.6 30.30 953.28 41.6 34.73 945.43
31.7 26.06 960.05 36.7 30.39 953 .13 41.7 34.82 945.26
31.8 26.15 959.92 36.8 30.48 952.98 41.8 34.91 945.09
31.9 26.23 959.79 36.9 30.56 952 .83 41.9 35.00 944.93
32.0 26.32 959.66 37.0 30.65 952.69 42.0 35.09 944.76

32.1 26.40 959.53 37.1 30.74 952.54 42.1 35.18 944.59
32.2 26 .49 959.40 37.2 30.83 952.39 42.2 35.27 944 .42
32.3 26 .57 959.27 37. 3 30.92 952.24 42.3 35.36 944.25
32.4 26.66 959.14 37.4 31.00 952.09 42.4 35.45 944.08
32.5 26.75 959.01 37.5 31.09 951.94 42.5 35.54 943 .91
32.6 26.83 958.87 37.6 31.18 951.79 42.6 35.63 943.74
32.7 26.92 958.74 37.7 31.27 951.63 42.7 35.72 943.57
32.8 27.00 958.61 37.8 31.35 951.48 42.8 35.81 943.40
32.9 27.09 958.47 37.9 31.44 951.33 42.9 35.90 943.23
38.0 31.53 951.18 43.0 35.99 943.06

33.1 27 .26 958.20 38.1 31.62 951.02 43.1 36.08 942.88
33.2 27.35 958.07 38.2 31.71 950.87 43 .2 36.17 942.71
33.3 27.44 957.94 38.3 31.79 950 .72 43.3 36.26 942.54
33.4 27.52 957.80 38.4 31.88 950.56 43 .4 36.35 942 .37
33.5 27.61 957.66 38.5 31 .97 950.41 43 .5 36.44 942.19
33.6 27.69 957.53 38.6 32.06 950.25 43.6 36.53 942.02
33.7 27.78 957.39 38.7 32.15 950 .10 43.7 36.62 941.84
33.8 27.87 957.26 38.8 32.24 949.94 43.8 36.71 941.67
33.9 27.95 957.12 38.9 32.32 949.79 43.9 36.80 941.49
34.0 28.04 956.98 39.0 32.41 949 .63 44.0 36.89 941.32
34.1 28.13 956.84 39.1 32.50 949.47 44.1 36.98 941.14
34.2 28 .21 956.70 39.2 32.59 949.32 44.2 37.07 940.97
34.3 28.30 956.57 39.3 32.68 949.16 44.3 37.16 940 .79
34.4 28.39 956.43 39.4 32.77 949.00 44.4 37.25 940.61
34.5 28.47 956.29 39.5 32.86 948 .84 44.5 37.35 940.43
34.6 28.56 956.15 39.6 32.94 948 .68 44.6 37.44 940.26
34.7 28.65 956.01 39.7 33.03 948.52 44.7 37.53 940.08
34.8 28.73 955.87 39.8 33.12 948.37 44.8 37.62 939 .90
34.9 28 .82 955.73 39.9 33.21 948.21 44.9 37.71 939.72
V-A670 Supplementary Chapter IV E 2014
% VIV % m/m Pzo (kg/m 3) % VIV % m/m P20 (kg/m 3) % VIV % m/m P20 (kg/m3 )
45 .0 37.80 939.54 50.0 42.43 930.14 55.0 47.18 919.96
45.1 37.89 939 .36 50.1 42.52 929.95 55.1 47.28 919 .75
45 .2 37.98 939.18 50.2 42.61 929 .75 55.2 47.38 919 .54
45.3 38.08 939.00 50.3 42.71 929.55 55.3 47.47 919 .33
45.4 38.17 938.82 50.4 42.80 929.35 55.4 47 .57 919.12
45.5 38.26 938.64 50.5 42.90 929.16 55.5 47 .67 918.91
45.6 38.35 938 .46 50.6 42.99 928.96 55.6 47.77 918 .69
45 .7 38.44 938.28 50.7 43.08 928.76 55.7 47.86 918 .48
45.8 38.53 938.10 50.8 43.18 928.56 55 .8 47.96 918.27
45 .9 38.62 937.91 50.9 43 .27 928.36 55.9 48.06 918.06
46 .0 38.72 937 .73 51.0 43.37 928.16 56.0 48 .15 917 .84
46.1 38.81 937.55 51.1 43.46 927 .96 56.1 48 .25 917.63
46.2 38.90 937.36 51.2 43.56 927 .77 56.2 48.35 917.42
46.3 38.99 937.18 51.3 43.65 927.57 56.3 48.45 917 .20
46.4 39.08 937 .00 51.4 43 .74 927 .36 56.4 48.54 916.99
46.5 39.18 936.81 51.5 43.84 927.16 56.5 48.64 916.77
46.6 39.27 936 .63 51.6 43.93 926.96 56.6 48.74 916.56
46.7 39.36 936.44 51.7 44.03 926.76 56.7 48.84 916.35
46.8 39.45 936.26 51.8 44.12 926.56 56.8 48.93 916 .l3
46 .9 39.54 936 .07 51.9 44.22 926.36 56.9 49.03 915.91
47.0 39.64 935.88 52.0 44.31 926.16 57.0 49.13 915.70

47.1 39.73 935.70 52.1 44.41 925.95 57.1 49.23 915.48
47 .2 39.82 935.51 52.2 44.50 925 .75 57. 2 49.32 915.27
47 .3 39.91 935.32 52.3 44.60 925.55 57.3 49 .42 915.05
47.4 40.00 935.14 52.4 44.69 925 .35 57.4 49 .52 914 .83
47.5 40.10 934.95 52.5 44.79 925.14 57.5 49.62 914.62
47.6 40.19 934.76 52.6 44.88 924.94 57.6 49.72 914.40
47 .7 40.28 934.57 52.7 44.98 924 .73 57.7 49.81 914.18
47 .8 40.37 934.38 52.8 45 .07 924.53 57.8 49.91 913.97
47 .9 40.47 934.19 52.9 45.17 924.32 57.9 50.01 913 .75
48.0 40.56 934.00 53.0 45.26 924.12 58.0 50.11 913 .53
48 .1 40 .65 9.33.81 53.1 45.36 923.91 58.1 50.21 913 .31
48.2 40.75 933.62 53.2 45.46 923 .71 58.2 50.31 913.09
48.3 40.84 933.43 53.3 45 .55 923.50 58.3 50.40 912.87
48.4 40.93 933.24 53.4 45.65 923.30 58.4 50.50 912 .65
48 .5 41.02 933 .05 53.5 45.74 923.09 58.5 50.60 912.43
48.6 41.12 932.86 53.6 45.84 922.88 58.6 50.70 912 .22
48.7 41.21 932.67 53.7 45.93 922.68 . 58.7 50.80 912.00
48 .8 41.30 932.47 53.8 46.03 922.47 58.8 50.90 911.78
48.9 41.40 932 .28 53.9 46.13 922.26 58.9 51.00 911.55
49 .0 41.49 932.09 54.0 46.22 922 .06 59.0 51.10 911.33

49.1 41.58 931.90 54.1 46.32 921.85 59.1 51.19 911.11
49.2 41.68 931.70 54.2 46.41 921.64 59.2 51.29 910 .89
49.3 41.77 931.51 54.3 46.51 921.43 59.3 51.39 910 .67
49 .4 41.86 931.31 54.4 46.61 921.22 59.4 51.49 910.45
49.5 41.96 931.12 54.5 46.70 921.01 59.5 51.59 910.23
49 .6 42.05 930.92 54.6 46.80 920 .80 59.6 51.69 910.01
49.7 42.14 930.73 54.7 46.90 920.59 59.7 51.79 909 .78
49 .8 42.24 930.53 54.8 46.99 920.38 59.8 51.89 909 .56
49 .9 42.33 930.34 54.9 47.09 920.17 59.9 51.99 909.34
% VIV % m/m P20 (kg/m 3) % VIV % m/m P20 (kg/m 3) % VIV % m/m P20 (kg/m
3
)
60.0 52.09 909.11 65.0 57.15 897.65 70 .0 62.39 885.56

60. 1 52.19 908.89 65.1 57.25 897.42 70.1 62.49 885.31
60.2 52.29 908 .67 65.2 57.36 897.18 70.2 62.60 885.06
60.3 52.39 908.44 65 .3 57.46 896.94 70.3 62.71 884.82
60.4 52.49 908.22 65.4 57.56 896.71 70.4 62 .81 884.57
60.5 52.59 908.00 65.5 57.67 896.47 70.5 62.92 884.32
60.6 52.69 907 .77 65 .6 57.77 896.23 70.6 63.03 884.07
60.7 52.79 907 .55 65.7 57.87 896.00 70 .7 63.13 883.82
60.8 52.89 907.32 65.8 57.98 895.76 70.8 63.24 883.57
60.9 52.99 907.10 65.9 58.08 895.52 70.9 63.35 883.32
61.0 53.09 906 .87 66.0 58.18 895.28 71.0 63.46 883.06
61.1 53.19 906.64 66.1 58.29 895.05 71.1 63.56 882.81
61.2 53.29 906.42 66.2 58.39 894.81 71.2 63.67 882.56
61.3 53.39 906.19 66.3 58.49 894.57 71.3 63.78 882 .31
61.4 53.49 905.97 66.4 58.60 894 .33 71.4 63.89 882.06
61.5 53.59 905 .74 66.5 58.70 894.09 71.5 63.99 881.81
61.6 53.69 905.51 66.6 58.81 893.85 71.6 64.10 881.55
61.7 53.79 905.29 66.7 58.91 893 .61 71.7 64.2 1 881.30
61.8 53.89 905.06 66.8 59.01 893 .37 71.8 64.32 881.05
61.9 53.99 904.83 66.9 59.12 893.13 71.9 64.43 880.79
62.0 54.09 904.60 67.0 59.22 892.89 72.0 64.53 880.54

62.1 54.19 904.37 67.1 59.33 892.65 72.1 64.64 880.29
62.2 54.30 904.15 67.2 59.43 892.41 72.2 64.75 880.03
62.3 54.40 903.92 67.3 59.54 892.17 72.3 64.86 879 .78
62.4 54.50 903.69 67.4 59.64 891.93 72.4 64.97 879.52
62.5 54.60 903.46 67.5 59.74 891.69 72.5 65.08 879.27
62.6 54.70 903.23 67.6 59.85 891.45 72.6 65.19 879.01
62.7 54.80 903.00 67.7 59.95 891.20 72.7 65 .29 878.75
62.8 54.90 902.77 67.8 60.06 890.96 72.8 65.40 878.50
62.9 55.00 902.54 67.9 60.16 890.72 72.9 65.51 878.24
63.0 55.11 902.31 68.0 60.27 890.48 73.0 65.62 877.99

63.1 55.21 902.08 68.1 60.37 890.23 73.1 65.73 877.73
63.2 55.31 901.85 68 .2 60.48 889.99 73.2 65.84 877.47
63 .3 55.41 901.62 68.3 60.58 889.75 73 .3 65.95 877.21
63.4 55.51 901.39 68.4 60.69 889.50 73.4 66.06 876 .96
63.5 55.61 901.15 68.5 60.80 889.26 73.5 66.17 876.70
63.6 55.72 900.92 68.6 60.90 889.01 73.6 66.28 876.44
63.7 55.82 900.69 68.7 61.01 888.77 73.7 66.39 876.18
63.8 55.92 900 .46 68.8 6l.l1 888 .52 73.8 66.50 875.92
63 .9 56.02 900 .23 68.9 61.22 888.28 73.9 66.61 875.66
64.0 56.12 899.99 69.0 61.32 888.03 74.0 66.72 875.40

64.1 56.23 899.76 69 .1 61.43 887 .79 74.1 66.83 875.14
64.2 56.33 899.53 69.2 61.54 887.54 74.2 66.94 874.88
64.3 56.43 899.29 69.3 61.64 887.29 74.3 67.05 874 .62
64.4 56.53 899 .06 69.4 61.75 887.05 74.4 67.16 874.36
64.5 56.64 898.83 69.5 61.85 886.80 74.5 67.27 874. 10
64.6 56.74 898.59 69.6 61.96 886.55 74.6 67.38 873.84
64.7 56.84 898.36 69.7 62.07 886 .31 74.7 67.49 873. 58
64. 8 56.94 898.12 69.8 62.17 886.06 74.8 67.60 873.32
64.9 57.05 897.89 69.9 62.28 885.81 74.9 67.71 873.06
V-A672 Supplementary Chapter IV E 2014
% VIV % mlm P20 (kg/m 3) % VIV % mlm P20 (kg/m 3) % VIV % mlm Pzo (kg/m 3)
75.0 67.82 872.79 80.0 73.48 859.27 85.0 79.40 844.85
75.1 67.93 872.53 80.1 73.60 858.99 85.1 79.53 844.55
75.2 68.04 872.27 80.2 73.71 858 .71 85.2 79.65 844.25
75 .3 68.15 872.00 80.3 73.83 858.43 85 .3 79.77 843.95
75.4 68.26 871.74 80.4 73.94 858.15 85.4 79.89 843 .65
75.5 68.38 87l.48 80.5 74.06 857.87 85.5 80.01 843.35
75.6 68.49 87l.21 80.6 74.18 857.59 85.6 80.14 843.05
75.7 68.60 870.95 80.7 74.29 857.3 1 85 .7 80.26 842 .75
75.8 68.71 870.68 80.8 74.41 857.03 85.8 80.38 842.44
75.9 68.82 870.42 80.9 74.53 856.75 85.9 80.50 842 .14
76.0 68.93 870. 15 81.0 74.64 856.46 86.0 80.63 84l.84

76.1 69.04 869.89 81.1 74 .76 856.18 86.1 80.75 841.53
76.2 69.16 869.62 81.2 74.88 855.90 86.2 80.87 84l.23
76.3 69.27 869.35 81.3 74.99 855.62 86.3 81.00 840.92
76.4 69.38 869.09 81.4 75.11 855.33 86.4 8l.l2 840.62
76.5 69.49 868 .82 8l.5 75.23 855.05 86.5 81.24 840.3 1
76.6 69.61 868.55 81.6 75.34 854.76 86.6 81.37 840.00
76.7 69.72 868.28 8 1.7 75.46 854.48 86.7 81.49 839.70
76.8 69.83 868 .02 8 1.8 75.58 854.19 86.8 81.61 839.39
76.9 69.94 867.75 81.9 75.70 853 .91 86.9 81.74 839.08
77.0 70.06 867.48 82.0 75.82 853.62 87.0 81.86 838.77

77.1 70.17 867 .21 82. 1 75 .93 853.34 87 .1 81.99 838.46
77.2 70.28 866.94 82.2 76.05 853.05 87.2 82.11 838.15
77.3 70.39 866.67 82.3 76.17 852.76 87.3 82.24 837.84
77.4 70.51 866.40 82.4 76.29 852 .48 87.4 82.36 837.52
77.5 70.62 866.13 82.5 76.41 852.19 87.5 82.49 837.21
77.6 70.73 865.86 82.6 76.52 851.90 87.6 82.61 836.90
77.7 70.85 865.59 82.7 76.64 851.61 87.7 82.74 836.59
77 .8 70.96 865.32 82.8 76.76 851.32 87 .8 82.86 836.27
77.9 71.07 865.05 82.9 76.88 85l.03 87.9 82.99 835.96
78.0 7l.l9 864.78 83.0 77.00 850.74 88.0 83. 11 835.64

78.1 71.30 864.50 83.1 77.12 850.45 88.1 83.24 835.32
78.2 7l.41 864.23 83.2 77.24 850.16 88.2 83.37 835.01
78.3 71.53 863.96 83.3 77.36 849.87 . 88 .3 83.49 834.69
78.4 71.64 863 .69 83.4 77.48 849.58 88.4 83.62 834 .37
78.5 71.76 863.41 83.5 77.60 849.29 88.5 83.74 834.05
78.6 71.87 863.14 83.6 77.72 848.99 88.6 83.87 833.73
78.7 71. 98 862.86 83.7 77.84 848.70 88.7 84.00 833.41
78.8 72.10 862.59 83.8 77.96 848 .41 88.8 84. 13 833.09
78.9 72.21 862.31 83.9 78.08 848. 11 88.9 84.25 832.77
79.0 72.33 862.04 84.0 78.20 847.82 89.0 84.38 832.45

79.1 72 .44 861.76 84.1 78.32 847.53 89. 1 84.51 832. 12
79.2 72.56 86l.49 84.2 78 .44 847 .23 89.2 84.64 831.80
79.3 72.67 86l.21 84.3 78.56 846.93 89.3 84.76 83l.48
79.4 72. 79 860.94 84.4 78.68 846.64 89.4 84.89 831.15
79.5 72.90 860.66 84.5 78.80 846.34 89.5 85.02 830.82
79.6 73.02 860.38 84.6 78.92 846.05 89.6 85 .15 830.50
79.7 73.13 860 .10 84.7 79.04 845.75 89 .7 85.28 830.17
79.8 73.25 859.83 84.8 79.16 845.45 89.8 85.4 1 829.84
79.9 73.36 859.55 84 .9 79.28 845. 15 89.9 85 .54 829.51
% VIV %mlrn P20 (kg/m3) % VIV %mlrn P20 (kg/m 3) % VIV %mlrn P20 (kg/m3)
90.0 85.66 829.18 94.0 91.01 815.18 98.0 96 .81 798 .90
90.1 85.79 828.85 94. 1 91.15 814.81 98.1 96.97 798.45
90.2 85.92 828.52 94.2 91 .29 814.43 98.2 97.12 798.00
90.3 86.05 828.19 94.3 91.43 814.06 98.3 97.28 797.54
90.4 86.18 827 .85 94.4 91.56 813 .68 98.4 97.43 797.08
90.5 86.31 827.52 94.5 91.70 813.30 98.5 97.59 796.62
90.6 86.44 827.18 94.6 91.84 812.92 98.6 97.74 796.15
90.7 86.57 826.85 94.7 91.98 812.54 98.7 97.90 795.68
90.8 86.71 826 .51 94.8 92.13 812.15 98 .8 98.06 795 .21
90.9 86.84 826.17 94.9 92.27 811.77 98.9 98.22 794.73
91.0 86.97 825.83 95.0 92.41 811.38 99.0 98 .38 794.25

91.1 87.10 825 .49 95.1 92.55 810.99 99.1 98.53 793 .77
91.2 87.23 825.15 95 .2 92.69 810.60 99.2 98.69 793.28
91.3 87.36 824.81 95.3 92.83 810.21 99.3 98.86 792.79
91.4 87.49 824.47 95.4 92.98 809.82 99.4 99.02 792.30
91.5 87.63 824.13 95.5 93.12 809.42 99 .5 99.18 791.80
91.6 87.76 823.78 95 .6 93.26 809.02 99.6 99.34 791.29
9l.7 87.89 823.44 95.7 93.41 808.63 99.7 99.50 790.79
91.8 88.02 823.09 95 .8 93.55 808.23 99.8 99.67 790.28
91.9 88.16 822.74 95.9 93.69 807.82 99 .9 99.83 789.76
92.0 88.29 822.39 96.0 93.84 807.42 100.0 100.0 789.24

92.1 88.42 822.04 96.1 93.98 807.01
92.2 88.56 821.69 96.2 94.13 806.61
92 .3 88.69 821.34 96.3 94.27 806.20
92.4 88.83 820.99 96.4 94.42 805 .78
92.5 88.96 820.63 96.5 94.57 805 .37
92.6 89.10 820.28 96.6 94.71 804.96
92.7 89.23 819.92 96.7 94.86 804.54
92 .8 89.37 819.57 96.8 95.01 804.12
92.9 89.50 819.21 96.9 95.16 803.70
93.0 89.64 818.85 97.0 95.31 803.27

93.1 89.77 818.49 97. 1 95.45 802.85
93.2 89.91 818.12 97.2 95 .60 802.42
93 .3 90.05 817.76 97.3 95 .75 801.99
93.4 90.18 817.40 97.4 95.90 801.55
93.5 90.32 817.03 97 .5 96.05 801.12
93.6 90.46 816.66 97.6 96.21 800.68
93 .7 90.59 816.30 97.7 96.36 800.24
93 .8 90.73 815.93 97.8 96.51 799.80
93.9 90.87 815.55 97.9 96.66 799.35
V-A674 Supplementary Chapter IV F 2014
medium, and Polyacrylamide rod gel electrophoresis, which

F. Pharmacopoeial Harmonisation are not within the scope of phannacopoeial harmonisation.
This general chapter is included for guidance of users. It provides The corresponding parts have been placed between black
infonnation on the degree of hannonisation of various general diamonds ( • • ).
chapters and monographs of the European Phannacopoeia and
The above differences in the Ph. Eur. text do not affect
those of the Japanese Phannacopoeia and United States
harmonisation as the general chapter provides additional
Phannacopeia. The chapter does not affect in any way the status
information .
of the monographs and general chapters as the authoritative
reference in any case of doubt or dispute where compliance with The texts of the 3 phannacopoeias are therefore considered
the European Phannacopoeia is required. harmonised.
The European Phannacopoeia Commission recognises the 2.2.47. Capillary electrophoresis
utility of working with other phannacopoeial bodies to
develop hannonised monographs and general chapters. Such As a result of an evaluation of the texts 4. Capülary
hannonisation is fully compatible with the declared aims of Electrophoresis in the Japanese Pharmacopoeia XV and
the Commission and has benefits of different kinds, notably < 1053> Biotechnology-derived anicles - Capillary
the simplification and rationalisation of quality control Electrophoresis in the United States Pharmacopeia USP33
methods and licensing procedures. Such hannonisation also NF28, and chapter 2.2.47. Capz7lary electrophoresis in the
enhances the benefits of the work of the International European Pharmacopoeia, the texts of the 3 phannacopoeias
Conference on Harmonisation (rCH) and the Veterinary are considered harmonised.
International Co-operation on Harmonisation (VICH) since
2.2.54. Isoelectric focusing
sorne of the guidelines developed depend on phannacopoeial
general chapters for their application. As a result of an evaluation of the texts 9. Isoelectric Focusing
Work on harmonisation is carried out by a well-defined but in the Japanese Pharmacopoeia XV and <1054>
Biotechnology-derived anicles - Isoelectric Focusing in the United
infonnal process in the Phannacopoeial Discussion Group
States Phannacopeia USP33 NF28, and chapter 2.2.54.
(PDG), in which the European Phannacopoeia, the Japanese
Isoelectric focusing in the European Pharmacopoeia, the texts
Phannacopoeia and the United States Phannacopeia are
of the 3 pharmacopoeias are considered harmonised.
associated. Information is given in this general chapter on
items that have been dealt with by the PDG. 2.2.55. Peptide mapping
Where hannonisation of general chapters is carried out, the
The following comparative commentary refers to the texts 15.
aim is to arrive at interchangeable methods or requirements
Peptide Mapping in the Japanese Pharmacopoeia XV and
so that demonstration of compliance using a general chapter
< 105 5> Biotechnology-derived A nicles - Peptide Mapping in
from one of the 3 phannacopoeias implies that the same
the United States Pharmacopeia USP31 NF26 2nd
result would be obtained using the general chapter of either
Supplement, and chapter 2.2.55. Peptide mapping in the
of the other phannacopoeias. When a formal declaration of
European Phannacopoeia.
interchangeability has been recommended by ICH, it will be
indicated in this general chapter. If residual differences Validation (USP) The USP has entitled this part System
remain in hannonised general chapters, infonnation is given Suitability. This terminology has been accepted by the 3
in this general chapter. pharmacopoeias.
Where hannonisation of monographs is carried out, the aim The use of peptide mapping for genetic stability
is to arrive at identical requirements for all attributes of a evaIuation (USP) This additional section does not impact
producto For sorne products, it can be extremely difficult to harmonisation since it is used only in development.
achieve complete hannonisation, for example because of The above differences in the USP text do not affect
differences in legal status and interpretation. It has therefore harmonisation.
appeared worthwhile to the PDG to approve and publish The texts of the 3 phannacopoeias are therefore considered
monographs in which as many attributes as possible are harmonised.
harmonised. Infonnation on any non-hannonised attributes is
included in this general chapter. 2.2.56. Amino acid analysis
The 3 phannacopoeias have undertaken not to make The following comparative commentary refers to the texts 1.
unilateral changes to harmonised monographs and general Amino Acid Analysis in the Japanese Pharmacopoeia XV and
chapters but rather to apply the agreed revision procedure < 1052 > Biotechnology-derived Anicles - Amino Acid Analysis
whereby all partners adopt a revision simultaneously. in the United States Pharmacopeia USP31 NF26 1st
Supplement, and chapter 2.2.56. Amino acid analysis in the
2.2.31. Electrophoresis European Pharmacopoeia.
The following comparative commentary refers to the texts 23. MethodoIogies of amino acid anaIysis: general
SDS-Polyacrylamide Gel Electrophoresis in the Japanese principIes (USP) The USP has replaced
Phannacopoeia XV and < 1056> Biotechnology-derived '6-aminoquinolyl-N-hydroxysuccinimidyl carbamate or
Anicles - Polyacrylamide Gel Electrophoresis in the United o-phthalaldehyde' with '6-aminoquinolyl-N-
States Phannacopeia USP31 NF26 2nd Supplement, and hydroxysuccinimidyl carbonate'.
chapter 2.2.31. Electrophoresis in the European
These reagents are different but compatible and the use of
Phannacopoeia.
one or the other does not affect harmonisation.
In the Ph. Eur. the hannonised chapter has been included as
The USP has added a detailed example to describe each
a section entitled Sodium dodecyl sulfate polyacrylamide gel
method listed below:
electrophoresis (SDS-PAGE), within a more general chapter
entitled Electrophoresis. The general chapter includes other - Method 1: post-column ninhydrin detection;
parts: General principie, Free or moving boundary - Method 2: post-column OPA derivatisation;
electrophoresis, Zone electrophoresis using a supporting - Method 3: pre-column PITC derivatisation;
2014 Supplementary Chapter IV F V-A675
- Method 4: pre-cotumn AQC derivatisation; 2.6.12. Microbiological examination of non-sterile

- Method 5: pre-column OPA derivatisation; products: microbial enumeration tests
- Method 6: pre-colurnn DABS-CI derivatisation; As a result of an evaluation of the texts 4.05 Microbiological
Examination of Non-sterile Products: 1. Microbiological
- Method 7: pre-column FMOC-Ct derivatisation;
Examination of Non-sterile Products - Microbial Enumeration
- Method 8: pre-column NBD-F derivatisation. Tests in the Japanese Pharmacopoeia XV 1SI Supplement and
The aboye examples are given for further information and do <6 1> Microbiological Examination of Non-stenle Products:
not affect harmonisation. Microbial Enwneration Tests in the United States
The texts of the 3 pharmacopoeias are therefore considered Pharmacopeia USP30 NF25, and chapter 2.6.12.
harmonised. Microbiological examination of non-sterile products: microbial
enumeration tests in the European Pharmacopoeia, the texts of
2.4.14. Sulfated ash the 3 pharmacopoeias are considered harmonised.
The following comparative commentary refers 10 the texts NOTE: ICH has declared this method interchangeable
2.44 Residue on Ignition Test in the Japanese Pharmacopoeia within the ICH regions.
XVand < 281 > Residue on Ignition in the United States
Pharmacopeia USP32 NF27 I Sl Supplement, and chapter 2.6.13. Microbiological examination ofnon-sterile
2.4.14. Sulfated ash in the European Pharmacopoeia. products: test for specified micro-organisms
The JP has added a non-harmonised introductory part, As a result of an evaluation of the texts 4.05 Microbiological
included between black diamonds, at the beginning of this Examination of Non-sterile Products: JI. Microbiological
chapter. It is given for further information and therefore does Examination of Non-sterile Products - Test for Specified
not affect harmonisation. Micro-organisms in the Japanese Pharmacopoeia XV 1sr
Supplement and < 62 > Microbiological Examination of Non-
The USP text allows for the test to be performed at an
sterile Products: Testfor Specified Micro-organisms in the United
ignition temperature other than 600 ± 50 oC if prescribed in
Sta tes Pharmacopeia USP30 NF25, and chapter 2.6.13.
an individual monograph. In the same way, a sample mas s
Microbiological examination of non-sterile products: test for
different from the usual quantity of 1-2 g can be used if
specified micro-organisms in the European Pharmacopoeia, the
prescribed in an individual monograph.
texts of the 3 pharmacopoeias are considered harmonised.
The USP has added a section, included between black
NOTE: ICH has declared this method interchangeable
diamonds, on the use of a muffte furnace and its calibration.
within the ICH regions.
The aboye differences in the USP text do not affect
harmonisation. 2.9.1. Disintegration oftablets and capsules
The texts of the 3 pharmacopoeias are therefore considered The following comparative commentary refers to the texts
harmonised. 6.09 Disintegration Test in the J apanese Pharmacopoeia XV
NOTE: ICH has declared this method interchangeable and < 701 > Disintegration in the United States
within the ICH regions. Pharmacopeia USP32 NF27 1SI Supplement, and chapter
2.9.1. Disintegration ofta~lets and capsules (Test A) in the
2.6.1. Sterility European Pharmacopoeia.
The following comparative commentary refers 10 the texts In the Ph. Eur. chapter, test A corresponds to the
4.06 Sterility Test in the partial revision of the J apanese harmonised chapter while test B does not and is intended for
Pharmacopoeia XV made official March 31, 2009, by the tablets and capsules that are greater than 18 mm long.
Ministry of Health, Labour and Welfare Ministerial Test B is not within the scope of pharmacopoeial
Notification No. 190 and < 71 > Sterility Tests in the United harmonisation and has been placed between black
Sta tes Pharmacopeia as presented in Pharmacopeial Forum, diamonds (+ +).
Volurne 34(6), Interim Revision Announcement No. The JP and USP specify procedures and acceptance criteria
6, December 1,2008, official on May 1, 2009, and chapter for different types of dosage forms . The equivalent
2.6.1. Stenlity in the European Pharmacopoeia. statements are included in the Ph. Eur. general monographs
The USP has added requirements that cover either pharmacy on dosage forms. These statements are not within the scope
bulk packages of antibiotics (which are not of concern in of pharmacopoeial harmonisation.
Europe and Japan) or medical devices (which are outside the In addition, the JP describes an auxiliary tube, and a metal
scope of the Ph. Eur. and JP). The corresponding parts, plate 10 secure the glass tubes. This has been placed between
which are not within the scope of pharmacopoeial black diamonds (+ +). The use of this tube and this plate
harmonisation, have been placed between black may have an impact on hydrodynamics and thus may affect
diamonds (+ +). harmonisation.
The JP has deleted the requirements for 'Catgut and other The texts of the 3 pharmacopoeias are therefore considered
surgical sutures for veterinary use' in Table 2, in the section harmonised.
'Direct inoculation of the culture medium' and in Table 3. NOTE: ICH has declared this method interchangeable
Catgut and other surgical sutures are outside the scope of the within the ICH regions subject to the conditions detailed
JP. below.
The aboye differences in the JP and USP texts do not affect For tablets and capsules larger than 18 mm long, for which a
harmonisation. different apparatus is used, the disintegration test is not
The texts of the 3 pharmacopoeias are therefore considered considered 10 be interchangeable in the 3 regions.
harmonised. The test for disintegration is not considered to be
NOTE: ICH has declared this method interchangeable interchangeable in the 3 regions for dosage forms referred to
within the ICH regions . in the pharmacopoeias as delayed-release, gastro-resistant or
enten·c-coated.
V-A676 Supplementary Chapter IV F 2014
2.9.7. Friability of uncoated tablets 2.9.26. Specific surface are a by gas adsorption
As a result of an evaluation of the texts 26. Tablet FI1'ability The following comparative commentary refers to the texts
Test in the Japanese Phannacopoeia XV and < 1216> Tablet 3.02 Specific SU/face Area by Gas Adsorption in the Japanese
Friability in the United States Phannacopeia USP31 Phannacopoeia XV and <846 > Specific SU/tace Area in the
NF26 1sI Supplement, and chapter 2.9.7. Friability of United States Phannacopeia USP31 NF26 1SI Supplement,
uncoated tablets in the European Phannacopoeia, the texts of and chapter 2.9.26. Specific sU/tace area by gas adsorption in
the 3 phannacopoeias are considered harmonised. the European Phannacopoeia.
The JP has chosen to express all the temperatures of this
2.9.17. Test for extractable volume ofparenteral chapter in degrees Celsius.
preparations
Multi-point measurement GP) The JP does not state
The following comparative commentary refers to the texts the meaning of the 22400 constant in the definition of the
6.05 Test fOI" Extractable Volume of Pal"enteral Preparations in specific surface are a S and does not require a test ro
the Japanese Pharmacopoeia XV and < 1> Injections in the detennine the linearity of the method.
United States Phannacopeia USP32 NF27 1SI Supplement,
Single-point measurement GP) The JP does not state
and chapter 2.9.17. Test for extractable volume of parenteral
the equivalent quantity of gas corresponding to the value of
preparations in the European Phannacopoeia.
PIPo, which is less precise CO.30) than in the other
The JP has added a non-hannonised introductory part, phannacopoeias CO.300).
ineluded between black diamonds, at the beginning of this
The JP do es not assume the material constant C to be
chapter. It is given for further information and does not
invariant.
affect harmonisation.
Measurements GP) The JP does not specify the
The USP has ineluded this test in general chapter < 1>
temperature required to perfonn the test for either method.
Injections, under a specific part entitled Detennination of
Volume of Injection in Containers. This does not affect The JP limits its volumetric method ro elassical instruments
harmonisation . and does not take alternative instruments into account.
The texts of the 3 phannacopoeias are therefore considered The 'above differences in the JP text might affect
harmonised. harrnonisation.
NOTE: ICH has deelared this method interchangeable Therefore only the texts of the Ph. Eur. and the USP are
within the ICH regions. considered harmonised.
2.9.19. Particulate contamination: sub-visible 2.9.36. Powder flow

particles The following comparative commentary refers to the texts 18.
The following comparative commentary refers to the texts Powder Flow in the Japanese Phannacopoeia XV and
6.07 Insoluble Particulate Matter Test for Injections in the < 1174> Powder Flow in the United States Phannacopeia
Japanese Phannacopoeia XV Ccorrected version dated USP31 NF26 1SI Supplement, and chapter 2.9.36. Powder
September 2007) and <788> Particulate Matter in Injections fiow in the European Phannacopoeia.
in the United States Phannacopeia USP32 NF27 2nd Flow through an orifice GP) The JP limits the use of
Supplement, and chapter 2.9.19. Particulate contamination: orifices ro elassical ones and does not allow vibrators or
sub-visible particles in the European Phannacopoeia. moving orifices. A test result using the JP method will be
The USP specifies that system suitability can be verified compatible with the Ph. Eur. and the USP. A Ph. Eur.
using USP Particle Count RS. This statement is not within the or USP test result wilI not comply with the JP when a
scope of phannacopoeial harmonisation. Ir has been placed vibrator or moving orifice is used.
between black diamonds C+ .) and it do es not affect
2.9.37. Optical microscopy
hannonisation.
The JP ineludes a detailed section on calibration of the As a result of an evaluation of the texts 3.04 Particle Size
apparatus. In particular, requirements for the quality of Determination in the Japanese Pharmacopoeia XV and
partiele-free water are given, which differ from those stated in <776> Optical Microscopy in the United States
Phannacopeia USP31 NF26 2nd Supplement, and chapter
the USP Csee section Reagents, Indicators and Solutions) and in
the Ph. Eur. Csee chapter 4.1.1). The section on calibration is 2.9.37. Optical microscopy in the European Phannacopoeia,
not within the scope of phannacopoeial harmonisation. It has the texts of the 3 phannacopoeias are considered
been placed between black diamonds C· .) and it does not harmonised.
affect harmonisation.
2.9.38. Particle-size distribution estimation by
In addition, the JP describes more stringent acceptance analytical sieving
criteria for parenteral preparations having a nominal volume
The following comparative commentary refers to the texts
of 100 mL. This was acknowledged by the PDG as a non-
3.04 Particle Size Detennination in the J apanese
hannonised item. Ir has been placed between black diamonds
Phannacopoeia XV and < 786 > Particle-size Distribution
C· .). The acceptance criteria for parenteral preparations
Detennination by Analytical Sieving in the United States
having a nominal volume of 100 mL are therefore considered
Phannacopeia USP31 NF26 1SI Supplement, and chapter
non-hannonised.
2.9.38. Particle-size distribution estimation by analytical sieving
The texts of the 3 phannacopoeias are therefore considered in the European Phannacopoeia.
harmonised except for the acceptance criteria for parenteral
Sieving methods - Dry sieving method GP) The JP
preparations having a nominal volume of 100 mL.
permits any powder on the down surface of the sieve to be
NOTE: ICH has deelared this method interchangeable brushed and combined with the fraction of the next sieve.
within the ICH regions except the acceptance criteria for
The abo ve difference in the JP text might affect
parenteral preparations having a nominal volume of 100 mL.
harmonisation.
2014 Supplementary Chapter IV F V-A677
Therefore only the texts of the Ph. Eur. and the USP are
considered harmonised.
5.1.4. Microbiological quality ofnon-sterile
pharmaceutical preparations and substances for
pharmaceutical use
The following comparative commentary refers to the texts 12.
Microbial Attributes 01 Non-sterile Pharmaceutical Products in the
Japanese Pharmacopoeia XV 1st Supplement and < 1111 >
Microbiological Attributes 01 Non-sterile Pharmaceutical Products
in the United States Pharmacopeia USP30 NF25, and
chapter 5.1.4. Microbiological quality 01 non-stenle
pharmaceutical preparations and substances 10r pharmaceutical use
in the European Pharmacopoeia.
A special Ph. Eur. provision for oral dosage forms containing
raw material s of natural origin is included within table
5.1.4.-1. AIso, a reference to chapter 5.1.8 giving
recommended acceptance criteria for the microbiological
quality of herbal medicinal products for oral use is included
in the textoThe corresponding parts, which are not within
the scope of pharmacopoeial harmonisation, have been
placed between black diamonds C* *).
The above differences in the Ph. Eur. text do not affect
harmonisation.
The texts of the 3 pharmacopoeias are therefore considered
harmonised.
NOTE: ICH has declared these texts interchangeable within
the ICH regions.
V-A678 Supplementary Chapter IV G 2014
G. Statistical Analysis 01 Results 01 Biological Assays and Tests

(Ph. Eur. General Text 5.3)
Contents
1. INTRODUCTION
1.1. General design and precision
2. RANDOMISATION AND INDEPENDENCE OF INDIVIDUAL TREATMENTS
3. ASSAYS DEPENDING UPON QUANTITATIVE RESPONSES
3.1. Statistical models
3.1.1. General principies
3.1.2. Routine assays
3.1.3. Calculations and restrictions
3.2. The parallel-line model
3.2.1. Introduction
3.2.2. Assay design
3.2.2.1. Completely randomised design
3.2.2.2. Randomised block design
3.2.2.3. Latin square design
3.2.2.4. Cross-over design
3.2.3. Analysis of variance
3.2.4. Tests of validity
3.2.5. Estimation of potency and confidence limits
3.2.6. Missing values
3.3. The slope-ratio model
3.3.1. Introduction
3.3.2. Assay design
3.3.3. Analysis of variance
3.3.3.1. The (líd + l)-design
3.3.3.2. The (hd)-design
3.3.4. Tests ofvalidity
3.3.5. Estimation of potency and confidence limits
3.3.5.1. The (hd + l )-design
3.3.5.2. The (hd)-design
3.4 Extended sigmoid dose-response curves
4. ASSAYS DEPENDING UPON QUANTAL RESPONSES
4.1. Introduction
4.2. The probit method
4.2.1. Tabulation of the results
4.2.2 . Tests ofvalidity
4.2 .3. Estimation of potency and confidence limits
4.2.4. Invalid assays
4.3. The logit method
4.4. Other shapes of the curve
4.5. The median effective dose
5. EXAMPLES
5.1. Parallel-line model
5.1.1. Two-dose multiple assay with completely randomised design
5.1.2. Three-dose Latin square design
5.1.3 . Four-dose randomised block design
5.1.4. Five-dose multiple assay with completely randomised design
5.1.5. Twin cross-over design
5.2. Slope-ratio model
5.2.1. A completely randomised (O,3,3)-design
5.2.2. A completely randomised (O,4,4,4)-design
2014 Supplementary Chapter IV G V-A679
5.3. Quantal responses

5.3.1. Pro bit analysis of a test preparation against a reference
5.3.2. Logit analysis and other types of analysis of a test preparation against a reference
5.3.3. The EDso detennination of a substance using the probit method
5.4. Extended sigmoid dose-response curves
5.4.1. Four-parameter logistic curve analysis
6. COMBINATION OF ASSAY RESULTS
6.1. Introduction
6.2. Weighted combination of assay results
6.2.1. Ca1culation of weighting coefficients
6.2.2. Homogeneity of potency estima tes
6.2.3. Ca1culation of the weighted mean and confidence limits
6.2.4. Weighted mean and confidence limits based on the intra- and inter-assay variation
6.3 . Unweighted combination of assay results
6.4. Example of a weighted mean potency with confidence limits
7. BEYOND THIS ANNEX
7.1. General linear models
7.2. Heterogeneity of variance
7.3 . Outliers and robust methods
7.4. Correlated errors
7.5. Extended non-linear dose-response curves
7.6 Non-parallelism of dose-response curves
8. TABLES AND GENERATING PROCEDURES
8.1. The F-distribution
8.2. The t-distribution
8.3. The X2-distribution
8.4. The <D-distribution
8.5. Random permutations
8.6. Latin squares
9. GLOSSARY OF SYMBOLS
10. LITERATURE
depends on the requirements set in the monograph for the

1. Introduction preparation concemed.
This chapter provides guidance for the design of bioassays The tenns "mean" and "standard deviation" are used here
prescribed in the European Phannacopoeia (Ph. Eur.) and as defined in most current textbooks of biometry.
for analysis of their results . It is intended for use by those
The tenns " stated potency" or "labelled potency", "assigned
whose primary training and responsibilities are not in
potency", "assumed potency", "potency ratio" and
statistics, but who have responsibility for analysis or
"estimated potency" are used in this section to indicate the
interpretation of the results of these assays, often without the
following concepts:
help and advice of a statistician. The methods of calculation
described in this annex are not mandatory for the bioassays - "stated potency" or "labelled potency": in the case of a
which themselves constitute a mandarory part of the Ph. Eur. formulated product a nominal value assigned from
Altemative methods can be used and may be accepted by the knowledge of the potency of the bulk material; in the case
competent authorities, provided thar they are supported by of bulk material the potency estimated by the
relevant data and justified during the assay validation process. manufacturer;
A wide range of computer sofrware is available and may be - "assigned potency": the potency of the standard
useful depending on the facilities available to, and the preparation;
expertise of, the analyst. - "assumed potency": the provisionally assigned potency of
Professional advice should be obtained in situations where: a a preparation ro be examined which forms the basis of
comprehensive treatment of design and analysis suitable for calculating the doses that would be equipotent with the
research or development of new products is required; doses to be used of the standard preparation;
the restrictions imposed on the assay design by this chapter - "potency ratio" of an unknown preparation; the ratio of
are not satisfied, for example particular laboratory constraints equipotent dos es of the standard preparation and the
may require cusromized assay designs, or equal numbers of unknown preparation under the conditions of the assay;
equally spaced doses may not be suitable; analysis is required - "estimated potency": the potency calculated from assay
for extended non-linear dose-response curves, for example as data.
may be encountered in irnmunoassays. An outline of
Section 9 (Glossary of symbols) is a tabulation of the more
extended dos e-response curve analysis for one widely used
important uses of symbols throughout this annex. Where the
model is nevertheless included in Section 3.4 and a simple
text refers ro a symbol not shown in this section or uses a
example is given in Section 5.4.
symbol to denote a different concept, this is defined in that
1.1. General design and precision part of the texto
Biological methods are described for the assay of certain
substances and preparations whose potency cannot be
adequately assured by chemical or physical analysis.
The principie applied wherever possible throughout these
2. Randomisation and independence 01
assays is that of comparison with a standard preparation so as individual treatments
to detennine how much of the substance ro be examined The allocation of the different treatments ro different
produces the same biological effect as a given quantity, the experimental units (animals, tubes, etc.) should be made by
Unit, of the standard preparation. It is an essential condition sorne strictly random process . Any other choice of
of such methods of biological assay that the tests on the experimental conditions that is not deliberately allowed for in
standard preparation and on the substance ro be examined the experimental design should also be made randomly.
be carried out at the same time and under identical Examples are the choice of positions for cages in a laboratory
conditions.
and the order in wruch treatrnents are administered.
For certain assays (detennination of virus titre for example) In particular, a group of animals receiving the same dose of
the potency of the test. sample is not expressed relative ro a any preparation should not be treated together (at the same
standard. This type of assay is dealt with in Section 4.5. time or in the same position) unless there is strong evidence
Any estimate of potency derived from a biological assay is that the relevant source of variation (for example, between
subject to random error due to the inherent variability of times, or berween positions) is negligible. Random allocations
biological responses and calculations of error should be may be obtained from computers by using the built-in
made, if possible, from the results of each assay, even when randomisation function. The analyst must check whether a
the oflicial method of assay is used. Methods for the design different series of numbers is produced every time the
of assays and the calculation of their errors are, therefore, function is started.
described below. In every case, before a statistical method is The preparations allocated ro each experimental unit should
adopted, a preliminary test is ro be carried out with an be as independent as possible. Within each experimental
appropriate number of assays, in order to ascertain the group, the dilutions allocated ro each treatrnent are not
applicability of this method. nonnally divisions of the same dose, but should be prepared
The confidence interval for the potency gives an indication of individually. Without this precaution, the variability inherent
the precision with which the potency has been estimated in in the preparation will not be fully represented in the
the assay. It is calculated with due regard ro the experimental experimental error variance. The result will be an under-
design and the sample size. The 95 per cent confidence estimation of the residual error leading ro:
interval is usually chosen in biological assays. Mathematical 1) an unjustified increase in the stringency of the test for
statistical methods are used ro calculate these Iimits so as to the analysis of variance (see Sections 3.2.3 and 3.2.4),
warrant the statement that there is a 95 per cent probability 2) an under-estimation of the true confidence limits for the
that these limits include the true potency. Whether this test, which, as shown in Section 3.2.5, are calculated
precision is acceptable to the European Phannacopoeia from the estimate of s2, the residual error mean square.
2014 Supplementary Chapter IV G V -A681
satisfactory. It can also improve the normality if the

3. Assays depending upon quantitative dlstribution is skewed to the right.
responses - The transformation of y to .,¡y is use fui when the
3.1. Statistical models observations follow a Poisson distribution i.e. when they
are obtained by counting.
3.1.1. General principIes
- The square transformation of y to y 2 can be useful if, for
The bioassays included in the Ph. Eur. have been conceived
example, the dose is more likely to be proponional to the
as "dilution assays", which means that the unknown
area of an inhibition zone rather than the measured
preparation to be assayed is supposed to contain the same
diameter of that zone.
active principie as the standard preparation, but in a different
ratio of active and inen components. In such a case the For sorne assays depending on quantitative responses, such
unknown preparation may in theory be derived from the as irnrnunoassays or cell-based in vitro assays, a large number
standard preparation by dilution with inen components. of doses is used. These doses give responses that completely
To check whether any particular assay may be regarded as a span the possible response range and produce an extended
dilution assay, it is necessary to compare the dose-response non-linear dose-response curve. Such curves are typical for
relationships of the standard and unknown preparations. aH bioassays, but for many assays the use of a large number
If these dose-response relationships differ significantly, then of doses is not ethical (for examp1e, in vivo assays) or
the theoretical dilution assay model is not valido Significant practical, and the aims of the assay may be achieved with a
differenees in the dos e-response relationships for the standard limited number of doses. Ir is therefore customary to restriet
and unknown preparations may suggest that one of the doses to that part of the dose-response range which is linear
preparations contains, in addition to the active principie, under suitable transformation, so that the methods of
other components which are not in en but which inftuence Sections 3.2 or 3.3 apply. However, in sorne cases analysis of
the measured responses. extended dose-response curves may be desirable. An outline
of one model which may be used for su eh analysis is given in
To make the effect of dilution in the theoretical model
Section 3.4 and a simple example is shown in Section 5.4.
apparent, it is useful to transform the dose-response
relationship to a linear function on the widest possible range There is another category of assays in which the response
of doses. 2 statistical models are of interest as models for the cannot be measured in each experimental unit, but in which
bioassays prescribed: the paralle1-line model and the slope- only the fraction of units responding to each treatment can
ratio model. be counted. This category is dealt with in Section 4.
The application of either is dependent on the fulfi1ment of 3.1.2. Routine assays
the following conditions: When an assay is in routine use, it is seldom possible to
1) the different treatments have been randomly assigned to check systematicaHy for conditions 1 to 3, because the
the experimental units; limited number of observations per assay is likely to inftuence
2) the responses to each treatment are normally distributed, the sensitivity of the statistical tests. Fortunately, statisticians
have shown that, in symmetrical balanced assays, small
3) the standard deviations of the responses within each
deviations from homogeneity of variance and normality do
treatment group of both standard and unknown
not seriously affect the assay results. The applieability of the
preparations do not differ significantly from 'one another.
statistical model needs to be questioned only if a series of
When an assay is being developed for use, the analyst has to assays shows doubtful validity. It may then be neeessary to
determine that the data collected from many assays meet perform a new series of preliminary investigations as
these theoretical conditions. discussed in Seetion 3.1.1.
- Condition 1 can be fulfilled by an efficient use of 2 other necessary conditions depend on the statistical model
Section 2. to be used:
- Condition 2 is an assumption which in practice is almost - for the paraHel-line model:
always fulfilled. Minor deviations from this assumption
4A) the relationship between the logarithm of the dose
will in general not introduce serious ftaws in the analysis
and the response can be represented by a straight
as long as several replicates per treatment are included.
line over the range of doses used,
In case of doubt, a test for deviations from normality
(e.g. the Shapiro-Wilk l test) may be performed. 5A) for any unknown preparation in the assay the
straight line is parallel to that for the standard.
- Condition 3 can be checked with a test for homogeneity
of variances (e.g. Bartlett's2 test, Cochran's3 test). - for the slope-ratio model:
Inspection of graphical representations of the data can 4B) the relationship between the dose and the response
also be very instructive for this purpose (see examples in can be represented by a straight line for each
Section 5). preparation in the assay over the range of doses
When conditions 2 and/or 3 are not met, a transformation of used,
the responses may bring a better fulfi1ment of these 5B) for any unknown preparation in the assay the
conditions. Examples are In y, .,¡y, y2 . straight line intersects the y-axis (at zero dose) at
- Logarithmic transformation of the responses y to In y can the same point as the straight line of the standard
be useful when the homogeneity of variances is not preparation (i.e. the response functions of all
preparations in the assay must have the same
intercept as the response function of the standard).
1 Wilk, M.B. and Shapiro, SS (1968). The joim assessmen! of nonnaliry
of severa! independem samples, T echnometrics 10, 825-839. Conditions 4A and 4B can be verified only in assays in which
2 Bartlen, M.S. (1937). Properties of sufficiency and statistical tests, Proc. at least 3 dilutions of each preparation have been tested.
Roy. Soco London, Series A 160, 280-282. The use of an assay with only 1 or 2 dilutions may be
3 Cochran, WG. (1951), Testing a linear re/aLion among variances,
Biometrics 7, 17-32.
justified when experience has shown that linearity and In general, the rejection of observations solely because a
parallelism or equal intercept are regularly fulfi1led. test for outliers is significant, is discouraged.
After having collected the results of an assay, and before - An exceptionally low residual error may once in a while
calculating the relative potency of each test sample, an occur and cause the F-ratios to exceed the critical values.
analysis of variance is performed, in order to check whether In such a case it may be justified to replace the residual
conditions 4A and 5A (or 4B and 5B) are fulfilled. For this, error estimated from the individual assay, by an average
the total sum of squares is subdivided into a certain number residual error based on historical data recorded in the
of sum of squares corresponding to each condition which has control charts.
to be fulfilled. The remaining sum of squares represents the 3.1.3. Calculations and restrictions
residual experimental error to which the absence or existence
According to general principies of good design the following
of the relevant sources of variation can be compared by a
3 restrictions are normally imposed on the assay designo They
series of F-ratios.
have advantages both for ease of computation and for
When validity is established, the potency of each unknown precision.
relative to the standard may be calculated and expressed as a
a) Each preparation in the assay must be tested with the
potency ratio or con verted to sorne unit relevant to the
same number of dilutions .
preparation under test e.g. an Intemational Unit. Confidence
limits may al so be estimated from each set of assay data. b) In the parallel-Iine model, the ratio of adjacent doses
must be constant for all treatrnents in the assay; in the
Assays based on the parallel-line model are discussed in
slope-ratio model, the interval between adjacent doses
Section 3.2 and those based on the slope-ratio model in
must be constant for all treatrnents in the assay.
Section 3.3.
c) There must be an equal number of experimental units to
If any of the 5 conditions (1, 2, 3, 4A, 5A or 1, 2, 3, 4B, 5B)
each treatrnent.
are not fulfilled, the methods of calculation described here
are invalid and an investigation of the assay technique should If a design is used which meets these restrictions, the
be made. calculations are simple. The formulae are given in Sections
The analyst should not adopt another transformation unless 3.2 and 3.3. It is recommended to use software which has
been developed for this special purpose. There are several
it is shown that non-fulfilment of the requirements is not
programs in existen ce which can easily deal with aH assay-
incidental but is due to a systematic change in the
designs described in the monographs. Not all programs may
experimental conditions. In this case, testing as described in
use the same formula e and algorithms, but they should aH
Section 3.1.1 should be repeated before a new transformation
lead to the same results.
is adopted for the routine assays.
Assay designs not meeting the aboye mentioned restrictions
Excess numbers of invalid assays due to non-parallelism or
may be both possible and correct, but the necessary formulae
non-linearity, in a routine assay carried out to compare
are too complicated to describe in this texto A brief
similar materials, are likely to refiect assay designs with
description of methods for calculation is given in Section 7.1.
inadequate replication. This inadequacy commonly results
These methods can also be used for the restricted designs, in
from incomplete recognition of all sources of variability
affecting the assay, which can result in underestimation of the which case they are equivalent with the simple formulae.
residual error leading to large F-ratios. The formulae for the restricted designs given in this text may
be used, for example, to create ad hoc programs in a
It is not always feasible to take account of all possible sources
of variation within one single assay (e.g. day-to-day spreadsheet. The examples in Section 5 can be used to c1arify
the statistics and to check whether such a program gives
variation) . In such a case, the confidence intervals from
correct results.
repeated assays on the same sample may not satisfactorily
overlap, and care should be exercised in the interpretation of
3.2. The paralle1-line mode1
the individual confidence intervals. In order to obtain a more
reliable estimate of the confidence interval it may be 3.2.1. Introduction
necessary to perform several independent assays and to The parallel-line model is illustrated in Figure 3.2.1.-1.
combine these into one single potency estimate and The logarithm of the doses are represented on the horizontal
confidence interval (see Section 6) . axis with the lowest concentration on the left and the highest
For the purpose of quality control of routine assays it is concentration on the right. The responses are indicated on
recommended to keep record of the estima tes of the slope of the vertical axis. The individual responses to each treatment
regression and of the estimate of the residual error in control are indicated with black dots. The 2 lines are the calculated
charts. In(dose)-response relationship for the standard and the
unknown.
- An exceptionally high residual error may indicate sorne
technical problem. This should be investigated and, if it Note: the natural logarithm (In or loge) is used throughout
can be made evident that something went wrong during this textoWherever the term "antilogarithm" is used, the
the assay procedure, the assay should be repeated. quantity eX is meant. However, the Briggs or "common"
An unusually high residual error may also indicate the logarithm (log or log¡o) can equally well be used. In this case
presence of an occasional outlying or aberrant observation. the corresponding antilogarithm is 10x.
A response that is questionable because of failure to For a satisfactory assay the assumed potency of the test
comply with the procedure during the course of an assay sample must be c10se to the true potency. On the basis of
is rejected. If an aberrant value is discovered after the this assumed potency and the assigned potency of the
responses have been recorded, but can then be traced to standard, equipotent dilutions (if feasible) are prepared,
assay irregularities, omission may be justified. i.e. corresponding doses of standard and unknown are
The arbitrary rejection or retention of an apparently expected to give the same response. If no information on the
aberrant response can be a serious source of bias. assumed potency is available, preliminary assays are carried
out over a wide range of doses to determine the range where 3.2.2.2. RANDOMISED BLOCK DESIGN
the curve is linear. In this design it is possible to segregate an identifiable source
of variation, such as the sensitivity variation between litters of
experimental animals or the variation between Petri dishes in
a diffusion microbiological assay. The design requires that
every treatment be applied an equal number of times in every
• Standard block (litter or Petri dish) and is suitable only when rhe block
• is large enough to accommodate all treatrnents once. This is
• Test illustrated in Secrion 5.1.3. Ir is also possible to use a
• sample randomised design with repetitions. The treatments should
be allocated randomly within each block. An algorithm to
obtain random permutarions is given in Section 8.5.
3.2 .2.3. LATIN SQUARE DESIGN
This design is appropriate when the response may be affected
by two different sources of variarion each of which can
assume k different levels or positions. For example, in a plate
assay of an antibiotic the treatments may be arranged in a
k x k array on a large plate, each rreatment occurring once
• in each row and each column. The design is suitable when
• the number of rows, the number of columns and the number
of treatrnents are equal. Responses are recorded in a square
formar known as a Latin square. Variarions due ro differences
in response among the k rows and among the k columns may
be segregated, thus reducing the error. An example of a Larín
Figure 3.2.1.-1. - The parallel-line model for a 3 + 3 assay
square design is given in Section 5.1.2 . An algorithm to
obtain Latin squares is given in Section 8.6. More complex
The more nearly correct the assumed potency of the designs in which one or more rrearments are replica red
unknown, the closer the 2 lines will be together, for they within the Latin square may be useful in sorne
should give equal responses at equal doses. The horizontal circumstances. The simplified formula e given in this Chapter
distance between the lines represents the "true" potency of are not appropriare for such designs, and professional advice
the unknown, relative to its assumed potency. The greater should be obrained.
the distance between the 2 lines, the poorer the assumed 3.2.2.4. CROSS-OVER DESIGN
potency of the unknown. If the line of the unknown is This design is useful when the experiment can be sub-
situated to the right of the standard, the assumed potency divided into blocks but ir is possible to apply only 2
was overestimated, and the calculations will indicate an treatments to each block. For example, a block may be a
estimated potency lower than the assumed potency. Similarly, single unir that can be tested on 2 occasions. The design is
if the line of the unknown is situated to the left of the intended to increase precision by eliminating the effects of
standard, the assumed potency was underestimated, and the differences between units while balancing the effect of any
calculations will indicate an estimated potency higher than difference between generallevels of response at the 2
the assumed potency. occasions. If 2 doses of a standard and of an unknown
3.2.2. Assay design preparation are tested, this is known as a twin cross-over test.
The following considerations will be useful in optimising the The experiment is divided into 2 parts separated by a
precision of the assay design: suitable time interval. Units are divided inro 4 groups and
1) the ratio between the slope and the residual error should each group receives 1 of the 4 treatments in the first part of
be as large as possible, the test. Units that received one preparation in the first part
of the test receive the other prepararíon on the second
2) the range of doses should be as large as possible,
occasion, and units receiving small doses in one part of the
3) the lines should be as close together as possible, test receive large doses in the other. The arrangement of
i.e. the assumed potency should be a good estimate of doses is shown in Table 3.2.2.-1. An example can be found
the true potency. in Section 5.1.5.
The allocation of experimental units (animals, tubes, etc.) to
different treatments may be made in various ways.
Table 3.2.2.-I. - Arrangemenl of doses in cross-over design
3.2 .2.1. COMPLETELY RANDOMISED DESIGN
If the totality of experimental units appears to be reasonably Group oí units Time I Time II
homogeneous with no indication that variability in response S, T2

will be smaller within certain recognisable sub-groups, the
allocation of the units to rhe different trearments should be 2 S2 T,
made randomly. 3 T, S,
If unirs in sub-groups such as physical posirions or
4 T2 SI
experimental days are likely to be more homogeneous than
the totality of the units, the precision of the assay may be
increased by introducing one or more restrictions into the
designo A careful distribution of the unirs over these 3.2.3. Analysis 01 variance
restrictions permirs irrelevant sources of variation ro be This section gives formulae that are required to carry out the
eliminated. analysis of variance and will be more easily undersrood by
V -A684 Supplememary Chapter IV G 2014
Table 3 2.3 -1 - Formulae for parallel-line assays with d doses of each preparation
Standard 1" Test sample 2 nd Test sample
(S) <n (U. etc.)
Mean response lowest

dose
S, T, UI
Mean response 2 nd dose 5, T, U,
Oo, ... ... ...
Mean response highest

Sd Td Ud
dose
Total preparation Ps = SI + S, + ... + Sd PT = TI + T, + ... + T d Pu = ... etc .
Linear contrast 1 1
Ls = lS1 + 2S, + ... +dSd - 2(d+1)Ps LT = 1Tl + 2T2 + ... + dTd - 2 (d + 1) PT Lu = .. .etc.
Table 3.2.3.-11. - Additional formulae for the construction of the analysis of uariance
n K = n (Ps + P T + ... )'
Hp
d hd
Table 3.2.3.-I1I. - Formulae to calculate the sum of squares and degrees of freedom
Source of variation Degrees of freedom (f) Sum of squares
Preparations h - 1 SSp<ep = H p (pJ + Pi. + ... ) - K

Linear regression 1 1 ,
SSreg = ¡;HL (Ls + LT + ... )
Non-parallelism h - 1 SSp", = HL (L1 + L~ + ... ) - SSreg
NOÍl-linearityl') h(d - 2) SSlin = SStreat - SSpr ep - SSreg - SSpar
Treatments hd - 1 SS'rea' = n (S~ + .. . + S~ + T¡ + .. . + TJ + ... ) - K
1'1Not calculated for twa-dose assays
Table 3.2.3.-IV. - Estimation of the residual error

Source of variation Degrees of freedom Sum of squares
Blocks (rows)l") n - 1 SSblock = hd (R~ + ... + R~) - K
¡
Columnsl ") n - 1 SScol = hd(C~ + ... + C~) - K
Completely randomised hd (n - 1) SSres = SStot - SStreat
Residual error""1 Randomised block (hd - 1) (n - 1) SSres = SStot - SStreat - SSblo ck
Latin square (hd - 2) (n - 1) SSre s = SStot - SStr eat - SSblock - SScol
Total nhd - 1 SSto' = ~ (y _ y)2
For Latin square designs, these formulae are only applicable if n = hd

1'1Not calculated for completely randomised designs
1"1 Ooly calculated for Latin square desigos
1'''1 Depends on the type of design
reference to the worked examples in Section 5.1. Reference the formulae can only be used if the doses are equally
should also be made to the glossary of symbols (Section 9). spaced, if equal numbers of treatments per preparation are
The formulae are appropriate for symmetrical assays where applied, and each treatment is applied an equal number of
one or more preparations to be examined (T, U, etc.) are times. It should not be attempted to use the formulae in any
compared with a standard preparation (S). It is stressed that other situation.
Apart from sorne adjusunents to the error term, the basic and the logarithm of the potency ratio of a test preparation,
analysis of data derived from an assay is the same for for example T, is:
completely randomised, randomised block and Latin square
designs . The formulae for cross-over tests do not entirely fit PT -Ps (3.2.5.-2)
this scheme and thes~ are incorporated into Examp\e 5.1.5. M!r db
Having considered the points discussed in Section 3.1 and
transformed the responses, if necessary, the values should be
averaged over each treatment and each preparation, as shown The calculated potency is an estimate of the "true potency"
in Table 3.2.3.-1. The linear contrasts, which relate to the of each unknown. Confidence limits may be calculated as the
slopes of the ln(dose)-response lines, should also be formed. antilogarithms of:
3 additional formulae, which are necessary for the
construction of the analysis of variance, are shown in CMir ± J(C - 1) (CM!} + 2V) (3 .2.5.-3)
Table 3.2.3.-11,
The total variation in response caused by the different
_ SSreg d V _ SSreg
where C - SS 2 2 an - b2 dn
treatments is now partitioned as shown in Table 3.2.3.-III reg - S t
the sums of squares being derived from the values obtained
in Tables 3.2.3.-1 and 3.2.3.-11. The sum of squares due to The value of t may be obtained from Table 8.2 for p = 0.05
non-lineariry can only be calculated if at least 3 doses per and degrees of freedom equal to the number of the degrees
preparation are inc\uded in the assay. of freedom of the residual error. The estimated potency (R T )
The residual error of the assay is obtained by subtracting the and associated confidence limits are obtained by multiplying
variations allowed for in the design from the total variation in the values obtained by AT after antilogarithms have been
response (Table 3.2.3.-IV). In this table ji represents the taken. If the stock solutions are not exactly equipotent on the
mean of all responses recorded in the assay. It should be basis of assigned and assurned potencies, a correction factor
noted that for a Latin square the number of replicate is necessary (Se e Examples 5.1.2 and 5.1.3) .
responses (n) is equal to the number of rows, columns or 3.2.6. Missing values
treatments (dh). In a balanced assay, an accident totally unconnected with the
The analysis of variance is now completed as follows. Each applied treatments may lead to the loss of one or more
surn of squares is divided by the corresponding number of responses, for example because an animal dies. If it is
degrees of freedom to give mean squares. The mean square considered that the accident is in no way connected with the
for each variable to be tested is now expressed as a ratio to composition of the preparation administered, the exact
the residual error (i) and the significance of these values ca\culations can still be performed but the formula e are
(known as F-ratios) are assessed by use ofTable 8.1 or a necessarily more complicated and can only be given within
suitable sub-routine of a computer programo the framework of general linear models (see Section 7.1).
3.2.4. Tests 01 validity However, there exists an approximate method which keeps
Assay results are said to be "statistically valid" if the outcome the simpliciry of the balanced design by replacing the missing
of the analysis of variance is as follows . response by a ca\culated value. The loss of information is
taken into account by diminishing the degrees of freedom for
1) The linear regression term is significant, the total sum of squares and for the residual error by the
i.e. the calculated probabiliry is less than 0.05. If this number of missing values and using one of the formulae
criterion is not met, it is not possible to calculate below for the missing values. It should be borne in mind that
95 per cent confidence limits. this is only an approximate method, and that the exact
2) The term for non-parallelism is not significant, method is to be preferred.
i.e. the ca\culated probabiliry is not less than 0.05. This If more than one observation is missing, the same formulae
indica tes th,at condition 5A, Section 3.1, is satisfied; can be used. The procedure is to make a rough guess at all
3) The term for non-lineariry is not significant, the missing values except one, and to use the proper formula
i.e. the ca\culated probabiliry is not less than 0.05 . This for this one, using all the remaining values inc\uding the
indicates that condition 4A, Section 3.1, is satisfied. rough guesses. Fill in the ca\culated value . Continue by
A significant deviation from parallelism in a multiple assay similarly ca\culating a value for the first rough guess. After
may be due to the inc\usion in the assay-design of a ca\culating all the missing values in this way the whole cyc\e
preparation to be examined that gives an ln(dose)-response is repeated from the beginning, each calculation using the
line with a slope different from those for the other most recent guessed or ca\culated value for every response to
preparations. Instead of dec\aring the whole assay invalid, it which the formula is being applied. This continues until 2
may then be decided to eliminate all data relating to that consecutive cyc\es give the same values; convergence is
preparation and to restart the analysis from the beginning. usually rapid.
When statistical validiry is established, potencies and Provided that the number of values replaced is small relative
confidence limits may be estimated by the methods described to the total number of observations in the full experiment
in the next section. (s ay les s than 5 per cent), the approximation implied in this
3.2.5. Estimation 01 potency and confidence limits replacement and reduction of degrees of freedom by the
nurnber of missing values so replaced is usually fairly
If 1 is the In of the ratio between adjacent doses of any
satisfactory. The analysis should be interpreted with great
preparation, the common slope (b) for assays with d doses of care however, especially if there is a preponderance of
each preparation is obtained from:
missing values in one treatment or block, and a biometrician
should be consulted if any unusual fearures are encountered.
b = HL (Ls + LT + ... ) (3.2.5.-1) Replacing missing values in a test without replication is a
Inh particularly delicate operation.
eompletely ral1domised design Unlike the parallel-line model, the doses are not transformed
In a completely randomised assay the missing value can be to logarithms.
replaced by the arithmetic mean of the other responses to the Just as in the case of an assay based on the parallel-line
same treatment. model, it is important that the assumed potency is close to
Ral1domised block design the true potency, and to prepare equipotent dilutions of the
The missing value is obtained using the equation: test preparations and the standard (if feasible). The more
nearly correct the assumed potency, the closer the 2 lines will
nB ' +kT' - G' be together. The ratio of the slopes represents the "true"
(3.2.6.-1) potency of the unknown, relative to its assumed potency.
(n - l)(k - 1)
If the slope of the unknown preparation is steeper than that
of the standard, the potency was underestimated and the
where B ' is the sum of the responses in the block containing calculations will indicate an estimated potency higher than
the missing value, T' the corresponding treatment total and the assumed potency. Similarly, if the slope of the unknown
G ' is the sum of all responses recorded in the assay. is less steep than that of the standard, the potency was
Latin square desigl1 overestimated and the calculations will result in an estimated
The missing value y ' is obtained from: potency lower than the assumed potency.
In setting up an experiment, all responses should be
k (B ' + e' + T') - 2G' (3.2.6.-2) examined for the fulfilment of the conditions 1, 2 and 3 in
(k - 1) (k - 2) Section 3.1. The analysis of variance to be performed in
routine is described in Section 3.3 .3 so that compliance with
where B ' and e' are the sums of the responses in the row conditions 4B and SB of Section 3.1 may be examined.
and column containing the missing value. In this case k = 11. 3.3.2. Assay design
eross-over design The use of the statistical analysis presented below imposes
If an accident leading to loss of values occurs in a cross-over the following restrictions on the assay:
design, a book on statistics should be consulted (e.g. D.]. a) the standard and the test preparations must be tested
Finney, see Section 10), because the appropriate formulae with the same number of equally spaced dilutions,
depend upon the particular treatment combinations. b) an extra group of experimental units receiving no
treatment may be tested (the blanks),
3.3. The slope-ratio model
e) there must be an equal number of experimental units to
3.3.1. Introduction
each treatment.
This model is suitable, for example, for sorne microbiological
As already remarked in Section 3.1.3, assay designs not
assays when the independent variable is the concentration of
meeting these restrictions may be both possible and correct,
an essential growth factor below the optimal concentration of
but the simple statistical analyses presented here are no
the medium. The slope-ratio model is illustrated in Figure
longer applicable and either expert advice should be sought
3.3.1.-1.
or suitable software should be used.
A design with 2 doses per preparation and 1 blank, the
• Standard "common zero (2h + 1)-design", is usually preferred, since it
gives the highest precision combined with the possibility to
check validity within the constraints mentioned aboye.
However, a linear relationship cannot always be assumed to
be valid down to zero-dose. With a slight loss of precision a
design without blanks may be adopted. In this case 3 doses
• per preparation, the "common zero (3h)-design", are
• Test preferred to 2 dos es per preparation. The doses are thus
• Sample
given as follows:
1) the standard is given in a high dose, near to but not
exceeding the highest dose giving a mean response on
• the straight portio n of the dose-response line,
• 2) the other doses are uniformly spaced between the
highest dose and zero dose,
3) the test preparations are given in corresponding doses
based on the assumed potency of the material.
A completely randomised, a randomised block or a Latin
dose x square design may be used, such as described in Section
3.2.2. The use of any of these designs necessitates an
Figure 3.3.1.·1. - The sZope-ratio modeZ for a 2 x 3 + 1 assay adjustment to the error sum of squares as described for
assays based on the parallel-line model. The analysis of an
The doses are represented on the horizontal axis with zero assay of one or more test preparations against a standard is
concentration on the left and the highest concentration on described below.
the right. The responses are indicated on the vertical axis. 3.3.3. Analysis of variance
The individual responses to each treatment are indicated with
3.3.3 .1. THE (HD + l )-DESIGN
black dots. The 2 lines are the calculated dos e-response
The responses are verified as described in Section 3.1 and, if
relationship for the standard and the unknown under the
necessary, transformed. The responses are then averaged over
assumption that they intersect each other at zero-dose .
Table 3.3.3. 1.-I. - Formulae for slope-ratio assays with d doses of each preparation and a blank
Standard 1st Test sample 2nd Test sample
(8) (1) (U, etc.)
Mean response lowes! dose s, T, u,

Mean response 2nd dose S2 T2 U2
Mean response. highest dose Sd Ud
Total preparation Ps = SI + S2 + ... + Sd PT = TI + T2 + ... + Td Pu =
Linear product Ls = lS1 + 2S2 + ... + dSd LT = 1T1 + 2T2 + ... + dTd Lu = ...
Intereept value as = (4d + 2) Ps - 6Ls aT = (4d + 2)PT - 6LT au = ...
Slope value bs = 2Ls - (d + 1) Ps bT = 2LT - (d + 1) PT bu =
Treatment value Gs = si + ... + S~ G T = T¡ + ... + TJ Gu =
Non-linearity("
J s = Gs _
p 2 _ _3b
~
2
_s_ J = GT _
p2
~
3b2
_ _ _T_ Ju = ...
T
d d3 - d d da - d
(') Not calculated for two-dose assays
Table 3.3.3.1.,". - Additional formulae for the construction of the analysis of variance
nhd 2 - nhd n as + aT +. K = n (B + P s + PT + ... )2
H ¡ = 4d3 _ 2d2 _ 2d a =
H B = hd2 _ hd + 4d + 2 h(d 2 - d) hd + 1
Table 3.3.3.1.-I1I. - Formulae to calculate the sum of squares and degrees of freedom
Source of variation Degrees of freedom (1) Sum of squares
Regression h SBreg = SStreat - SSblank - SSint - SSlin
Blanks 1 SSbl.nk = HB (B - a)2
Intersection h - 1 SSin' = H¡ ((a~ + a~ + ... ) - h (d 2 _ d)2 a 2 )

Non,linearity(') h(d - 2) SSlin = n (Js + J T + ... )
2
Treatments hd SS,,".t =n (B + G s + GT + ... ) - K
(" Not calculated for two-dose assays
Table 3.3.3.1.-IV. - Estimation of the residual error
Souree of variation Degrees of freedom Sum of squares
Blocks (rows)") n - 1 SSblo ck = hd (Ri + ... + R~) - K
¡
Columns'''' n - 1 SScol = hd (c¡ + ... + C~) - K
Completely
randomised
(hd + l )(n - 1) SSre3 = SStot - SStreat
Residual error"'"
Randomised block hd(n - 1) SS res = SStot - SStreat - SSblock
Latin square (hd - 1) (n - 1) SSres = SSt ot - SStreat - SSblock - SScol
Total nhd +n - 1 SS'o' = 2: (y - y)2
For Latin square designs, Ihese formulae are only applicable if n = hd

") Nol calculated for completely randomised designs
,.. , Only calculated for Latin square designs
,''') Depends on the type of design
each treatment and each preparation as shown in

Table 3.3.3.1.-1. Additionally, the mean response for blanks a' = (2dh +(2d1) -B 3)+ +(2d2d- +3)1 ha (3.3.5.1.-1)
(B) is calculated.
The sums of squares in the analysis of variance are calculated The slope of the standard, and similarly for each of the other
as shown.in Tables 3.3.3.1.-I to 3.3.3.1.-III. The sum of preparations, is calculated from:
squares due to non-linearity can only be calculated if at least
3 doses of each preparation have been inc1uded in the assay.
The residual error is obtained by subtracting the variations 6Ls - 3d(d + 1) a'
b~ = --~~~~--~-
3
2d2
+ 3d + d (3.3.5.1.-2)
allowed for in the design from the total variation in response
(Table 3.3.3.1.-N) .
The analysis of varüince is now completed as follows. Each The potency ratio of each of the test preparations can now
sum of squares is divided by the corresponding number of be calculated from:
degrees of freedom to give mean squares. The mean square
for each variable to be tested is now expressed as a ratio to
the residual error (S2) and the significance of these values
by
b'S (3.3.5.1.-3)
(known as F-ratios) are assessed by use of Table 8.1 or a
suitable sub-routine of a computer programo
3.3.3.2. THE (HD)-DESIGN which has to be multiplied by A T , the assumed potency of
The formulae are basically the same as those for the (hd + the test preparation, in order to find the estimated potency
l )-design, but there are some slight differences. R T . If the step between adjacent doses was not identical for
- B is discarded from all forrnulae. the standard and the test preparation, the potency has to be
multiplied by Is/h. Note that, unlike the parallel-line
analysis, no antilogarithms are calculated.
K = n(Ps+PT+ ... )2 The confidence interval for RT' is ca1culated from:
hd
CRfr - K' ± J(C - 1) (CR7 + 1) + K' (K' - 2CRfr)
- SSblank is removed from the analysis of variance.
- The number of degrees of freedom for treatments (3.3.5.1.-4)
becomes hd - l.
b'2
- The number of degrees of freedom of the residual error where C = b'2 52 2V and K' = (C - 1) V2
and the total variance is calculated as described for the 5 - s t 1
parallel-line model (see Table 3.2.3.-IV).
Validity of the assay, potency and confidence interval are VI are Vz are related to the variance and covariance of the
found as described in Sections 3.3.4 and 3.3.5. numerator and denominator of R T . They can be obtained
3.3.4. Tests o/ validity from :
Assay results are said to be "statistically valid" if the outcome
of the analysis of variance is as follows:
1) the variatíon due to blanks in (hd + 1)-designs is not
v-
I -
6 ( 1+
n (2d + 1) d (d 1)
3)
+ 2 (2d + 1) + hd (d - 1)
significant, i.e. the calculated probabílity is not smaller
(3.3.5.1.-5)
than 0.05. This indicates that the responses of the
blanks do not significantly differ from the common
intercept and the linear relationship is val id down to zero
dose;
2) the variatíon due to intersection is not significant,
V
2
= 3d(d + 1)
(3d + 1) (d + 2) + hd (d - 1) (3.3.5.1.-6)
í.e. the calculated probability is not less than 0.05. This
indicates that condition 5B, Section 3.1 is satisfied;
3) in assays inc1uding at least 3 doses per preparation, the The confidence limits are multiplied by A T , and if necessary
variation due to non-linearity is nor significant, by Is/h,
i.e. the calculated probability is not less than 0.05. This 3.3.5.2. THE (HD)-DESIGN
indicates that condition 4B, Section 3.1 is satisfied. The formulae are the same as for the (hd + 1)-design, with
A significant variation due to blanks indicates that the the following modifications:
hypothesis of linearity is not valid near zero dose. If this is
likely to be systematic rather than incidental for the type of
assay, the (hd-design) is more appropriate. Any response to
a' = a (3.3.5.2.-1)
blanks should then be disregarded.
When these tests indica te that the assay is valid, the potency
is calculated with its confidence limits as described in Section
3.3.5. VI = nd (2d + 1)
6 (' +1 + 3) d 1 h (d - 1) (3.3.5.2.-2)
3.3.5. Estimation o/ potency and confidence limits
3.3.5.1. THE (HD + l )-DESIGN
The common intersection a' of the preparations can be 3 (d + 1)
calculated from: (3.3.5.2.-3)
3 (d + 1) + h (d - 1)
3.4. Extended sigmoid dose-response curves upper and lower asymptotes and slopes, then one or all of
This model is suitable, for example, for sorne irnmunoassays these conditions may not be satisfied.
when analysis is required of extended sigmoid dose-response The logis tic model raises a number of statistical problems
curves. This model is illustrated in Figure 3.4.-1. which may require different solutions for different types of
assays, and no simple summary is possible. A wide variety of
possible approaches is described in the relevant literature.
Professional advice is therefore recommended for this type of
analysis. A simple example is nevertheless included in Section
• • 5.4 to illustrate a "possible" way to analyse the data
• • presented. A short discussion of altemative approaches and
other statistical considerations is given in Section 7.5.
If professional advice or suitable software is not available,
altemative approaches are possible: 1) if "reasonable"
estimates of the upper limit (ex) and lower limit (8) are
available, select for aH preparations the doses with mean of
the responses (u) falling between approximately 20 per cent
and 80 per cent of the limits, transform responses of the
Test sample
selected doses to y
u --
= In ( -
a-u
8) and use the parallel line
model (Section 3.2) for the analysis; 2) select a range of
• • doses for which the responses (u) or suitably transformed
• responses, for example In(u), are approximately linear when
•
plotted against In(dose); the parallelline model (Section 3.2)
may then be used for analysis.
In dose (x)
Figure 3.4.-1. - The four-parameter logis tic curve model

4. Assays depending upon quantal
The logarithms of the doses are represented on the horizontal
responses
axis with the lowest concentration on the left and the highest 4.1. Introduction
concentration on the right. The responses are indicated on In certain assays it is impossible or excessively laborious to
the vertical axis. The individual responses to each treatment measure the effect on each experimental unit on a
are indicated with black dots. The 2 curves are the ca1culated quantitative scale. Instead, an effect such as death or
ln(dose)-response relationship for the standard and the test hypoglycaemic symptoms may be observed as either
preparation. occurring or not occurring in each unit, and the result
The general shape of the curves can usually be described by depends on the number of units in which it occurs. Such
a logistic function but other shapes are also possible. Each assays are called quantal or all-or-none.
curve can be characterised by 4 parameters: The upper The situation is very similar to that described for quantitative
asymptote (ex), the lower asymptote (8), the slope-factor (~), assays in Section 3.1, but in place of n separate responses to
and the horizontallocation (y). This model is therefore often each treatrnent a single value is recorded, i.e. the fraction of
referred to as a four-parameter mode!. A mathematical units in each treatment group showing a response. When
representation of the ln(dose)-response curve is: these fractions are pIotted against the logarithms of the dos es
the resulting curve will tend to be sigmoid (S-shaped) rather
a-8 than linear. A mathematical function that represents this
u - 8+---::-;- -.,.
- 1 + e-{3(x - -y) sigmoid curvature is used to estimate the dose-response
curve. The most commonly used function is the cumuIative
normal distribution function. This function has sorne
For a val id assay it is necessary that the curves of the theoretical merit, and is perhaps the best choice if the
standard and the test preparations have the same slope- response is a reftection of the tolerance of the units. If the
factor, and the same maximum and minimum response level response is more likely to depend upon a process of growth,
at the extreme parts. Only the horizontallocation (y) of the the logistic distribution model is preferred, although the
curves may be different. The horizontal distance between the difference in outcome between the 2 models is usually very
curves is related to the "true" potency of the unknown. If the small .
assay is used routinely, it may be sufficient to test the
The maximum likelihood estimators of the slope and Iocation
condition of equal upper and lower response levels when the
of the curves can be found onIy by applying an iterative
assay is developed, and then to retest this condition directly
procedure. There are many procedures which lead to the
only at suitable intervals or when there are changes in
same outcome, but they differ in efficiency due to the speed
materials or assay conditions.
of convergence. One of the most rapid methods is direct
The maximum-likelihood estimates of the parameters and optimisation of the maximum-likeIihood function (se e
their confidence intervals can be obtained with suitable Section 7.1), which can easily be performed with computer
computer programs. These computer programs may include programs having a built-in procedure for this purpose.
sorne statistical tests refiecting validity. For example, if the Unfortunately, most of these procedures do not yield an
maximum likelihood estimation shows significant deviations estimate of the confidence interval, and the technique to
from the fitted model under the assumed conditions of equal obtain it is too complicated to describe here. The technique
Table 4.2.1.-1. - First working table

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
dose n r x p y ip Z y w wx wy wx' wy' wxy
E= E= E= E= E= E=
T
E= E= E= E= E= E=
etc.
described below is not the most rapid, but has been chosen (3) the number of units r giving a positive response to the
for its simplicity compared to the altematives. It can be used treatment,
for assays in which one or more test preparations are (4) the logarithm x of the dose,
compared to a standard. Furthermore, the following
(5) the fraetion p = rln of positive responses per group.
conditions must be fulfilled:
The first cycle starts here.
1) the relationship between the logarithm of the dose and
the response can be represented by a cumulative normal (6) column Y is filled with zeros at the first iteration,
distribution curve, (7) the corresponding value <1> = <I>(Y) of the cumulative
2) the curves for the standard and the test preparation are standard normal distribution function (see also
parallel, i.e. they are identically shaped and may only Table 8.4).
differ in their horizontal location, The columns (8) to (10) are calculated with the following
3) in theory, there is no natural response to extremely low formulae:
doses and no natural non-response to extremely high
doses. _y2/2
(8) z=_e__ (4.2.1.-1)
4.2. The probit method v'27T
The sigmoid eurve ean be made linear by replacing eaeh
response, i.e. the fraetion of positive responses per group, by
the corresponding value of the cumulative standard normal (9). p-4> (4.2.1.-2)
y=Y+--
distribution. This value, often referred to as "normit", ranges z
theoretically from - CIJ to + CIJ. In the past it was proposed
to add 5 to each normit to obtain "probits" . This faeilitated
the hand-performed ealculations beeause negative values were
avoided. With the arrival of eomputers the need to add 5 to (10) (4.2.1.-3)
the normits has disappeared. The term "normit method"
would therefore be better for the method described below.
However, since the term "pro bit analysis" is so widely The columns (11) to (15) ean easily be calculated from
spread, the term will, for historical reasons, be maintained in columns (4), (9) and (10) as wx, wy, wx2, wy2 and w.xy
this texto respectively, and the sum (L) of each of the columns (10) to
(15) is calculated separately for eaeh of the preparations.
Once the responses have been linearised, it should be
possible to apply the parallel-line analysis as described in The sums ealculated in Table 4.2.1.-1 are transferred to
Section 3.2. Unfortunately, the validity condition of eolumns (1) to (6) ofTable 4.2.1.-II and 6 additional
homogeneity of variance for each dose is not fulfilled. columns (7) to (12) are calculated as follows:
The variance is minimal at normit = O and increases for
positive and negative values of the normit. It is therefore (¿WX)2
neeessary to give more weight to responses in the middle part
(7) Sxx = ¿wx2 - ~ (4.2.1.-4)
of the curve, and les s weight to the more extreme parts of the
curve. This method, the analysis of variance, and the
estimation of the potency and eonfidence interval are
described below. _ '" (¿wx)(¿wy)
(8) S xy - ~ wxy - I: w (4.2.1.-5)
4.2.1. Tabulation ofthe results
Table 4.2 .1.-1 is used to enter the data into the columns
indicated by numbers:
(1) the dose of the standard or the test preparation, (9) (4.2.1.-6)
(2) the number n of units submitted to that treatment,
Table 4.2.1.-11. - Second working table

(1) (2) (3) (4) (5) (6) (7) (8) (9) (lO) (11) (l2)
:¿;w :¿;wx :¿;wy :¿;wi' :¿;w!l :¿;wxy Su SXY S" x Ji a
S
T
etc.
:¿;= :¿;=
(10) (4.2.1.-7) (4.2.3.-1)
and the antilogarithm is taken. Now let t = 1.96 and s = 1.

Confidence limits are calculated as the antilogarithms of:
(11) (4.2.1.-8)
CM!¡.-(C -l )(XS - XT)±V(C - l) (v ¿ S xx +C(M!¡' - XS + XT )2)

The common slope b can now be obtained as:
(4.2.3.-2)
b =~ (4.2.1.-9) b ¿ Sxx
2
1 1
~ where e = b2 ¿ S xx _ s 2 t 2 and V =-
¿-w+ -
¿w-
S T
and the intercept a of the standard, and similarly for the test
preparations is obtained as: 4.2.4. Invalid assays
If the test for deviations from linearity described in Section
(12) a=fi-bx (4.2.1.-10) 4.2.2 is significant, the assay should normally be rejected.
If there are reasons to retain the assay, the formulae are
Column (6) of the first working table can now be replaced by slightly modified. t becomes the t-value (p = 0.05) with the
y = a + bx and the cycle is repeated until the difference same number of degrees of freedom as used in the check for
between 2 cycles has become small (e.g. the maximum linearity and i becomes the l value divided by the same
difference of Y between 2 consecutive cycles is smaller than number of degrees of freedom (and thus typically is greater
10- 8). than 1).
The test for parallelism is also slightly modified. The l value
4.2.2. Tests 01 validity
for non-parallelism is divided by its number of degrees of
Before calculating the potencies and confidence intervals,
freedom. The resulting value is divided by i calculated
validity of the assay must be assessed. If at least 3 doses for
aboye to obtain an F-ratio with h - 1 and N - 2h degrees of
each preparation have been included, the deviations from
freedom, which is evaluated in the usual way at the 0.05
linearity can be measured as follows: add a 13 th column to
significan ce leve!.
Table 4.2.1.-II and fill it with:
4.3. The logit method
(4.2.2.-1 ) As indicated in Section 4.1 the logit method may sometimes
be more appropriate. The name of the method is derived
from the logit function which is the inverse of the logistic
The column total is a measure of deviations from linearity distribution. The procedure is similar to that described for
and is approximately X2 distributed with degrees of freedom the pro bit method with the following modifications in the
equal to N - 2h. Significance of this value may be assessed formula e for <1> and Z.
with the aid of Table 8.3 or a suitable sub-routine in a
computer programo If the value is significant at the 0.05 1 (4.3.-1)
<I>
probability level, the assay must probably be rejected (see 1 + e-Y
Section 4 .2.4).
When the aboye test gives no indication of significant
deviations from linear regression, the deviations from e- Y (4.3.-2)
parallelism are tested at the 0.05 significance level with: Z
{1 +e - y )2
(4.2.2.-2)
4.4. Other shapes of the curve
with h - 1 degrees of freedom . The probit and logit method are almost always adequate for
the analysis of quantal responses called for in the European
4.2.3. Estimation 01 potency and confidence limits Pharmacopoeia. However, if it can be m ade evident that the
When there are no indications for a significant departure In(dose)-response curve has another shape than the 2 curves
from parallelism and linearity the In(potency ratio) M'T is described abo ve, another curve <1> may be adopted. Z is then
calculated as: taken to be the first derivative of <1>.
For example, if it can be shown that the curve is not Tabie 5. 1. 1.-I. - Response metameter y : mass of ascorbic acid
symmetric, the Gompertz distribution may be appropriate (mg) per 100 g of adren al gland
(the gompit method) in which case Standard S Preparation T Preparation U
Y Y S, S, T, T2 U, U2
<l> = 1- e - e andZ = eY-e •
300 289 310 230 250 236
4.5. The median 'effective dose 310 221 290 210 268 213
In sorne types of assay it is desirable to determine a median 330 267 360 280 273 283
effective dose which is the dose that produces a response in
50 per cent of the units. The pro bit method can be used ro 290 236 341 261 240 269
determine this median effective dose (ED so ), but since there 364 250 321 241 307 251
is no need to express this dose relative ro a standard, the 328 231 370 290 270 294
formulae are slightly different.
390 229 303 223 317 223
Note: a standard can optionally be included in order ro
validate the assay. Usually the assay is considered valid if the 360 269 334 254 312 250
calculated ED50 of the standard is close enough to the 342 233 295 216 320 216
assigned ED50 . What " close enough" in this context means
306 259 315 235 265 265
depends on the requirements specified in the monograph.
The tabulation of the responses ro the test samples, and Mean 332.0 248.4 323.9 244.0 282.2 250.0
optionallya standard, is as described in Section 4.2.1.

The test for linearity is as described in Section 4.2 .2. A test
for parallelism is not necessary for this type of assay.
The EDso of test sample T, and similarly for the other 400
samples, is obtained as described in Section 4.2.3, with the •

380
following modifications in formulae 4.2.3.-1 and 4.2.3.-2) . •
360 •• •
- aT (4.5 .-1) • •
MIr b
;.., 340

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2014

Infrared Reference Spectra

Preparation of Infrared Reference Spectra

AH spectra presented in this section were recorded using either a

Polystyrene Instrument: Dispersive Phase: Thin Film Thickness : O.038mm

g 20 · t~ ________ '·_··~~ __·~"··~ 1

Polystyrene Instrumenl: Fourier Transform Phase: Thin Film Thickness: O.038mm

Abacavir RS464 Instrumenl: Fourier Transform Phase: Potassium Bromide Disc

Acenoco um arol RSOOJ Instrument: Dispersive Phase: Potassium Bromide Disc

Acctazolamide RS002 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Acetylcysteíne RS003 Instrument: Dispersive Phase: Potassium Bromide Disc

Adrenalíne (Epínephríne) RS004 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Alfuzosin RS446 Instrument: Fourier Transform Phase: Potassium Chloride Disc

Amantadine RS006 Inslrument: Dispersive Phase: Polassium Bromide Disc

Aminoglutethimide RS403 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

Amisulpride RS462 Instrumenl: Fourier Transform Phase: Attenuated Total Reflectance

Amoxicillin Sodium RSOIO Instrumenl: Dispersive Phase: Potassium Bromide Disc

Amoxicillin Trihydrate RSOll Instrumenl: Dispersive Phase : Potassium Bromide Disc

2000,0 1800 1600 1400 1200 1000 800 600 400,0

Ampicillin Sodium RS366 Instrument: Dispersive Phase: Potassium Bromide Disc

Ampicillin Trihydrate RS013 Instrument: Dispersive Phase: Potassium Bromide Disc

Amylmetacresol RS014 Instrument: Dispersive Phase : Thin Film

Atenolol RS015 Instrument: Dispersive Phase: Potassium Bromide Disc

Azapropazone RSOl6 Instrument: Dispersive Phase: Potassium Bromide Disc

Anhydrous Azapropazone RS017 Instrument: Dispersive Phase: Potassium Bromide Disc

1800 1600 1400 1200 1000 800 600 400.0

Benorilate RS023 Instrumenl: Dispersive Phase: Potassium Bromide Disc

Benzatropine Mesilate RS026 Instrumenl: Dispersive Phase: Liquid Paraffin Mull

Benzoic Acid RS025 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Betamethasone RS029 Inslrument: Dispersive Phase: Polassium Bromide Disc

Bezafibrate RS419 Inslrument: Fourier Transform Phase: Polassium Bromide Disc

Bronopol RS03 1 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Buclizine Hydrochloride RS032 Instrument: Dispersive Phase: Potassium Ch loride Disc

Bumetanide RS033 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Bupivacaine RS034 Instrument: Dispersive Phase : Liquid Paraffin Mull

1600 1400 1200 1000 800 600 400.0

Busulfan RS035 Instrumenl: Dispersive Phase: Potassium Bromide Disc

Butyl Hydroxybenzoate RS036 Instrumenl: Dispersive Phase: Potassium Bromide Disc

40 +. I .... -...... "~'"f-''~'~'''~''' ... _- '1

~ 20 .¡. ___ ..•....__,. -1- --

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Captopril RS038 Instrument: Dispersive Phase: Potassium Bromide Disc

Carbamazepine RS406 Instrument: Fourier Transform Phase: Potassium Bromide Disc

Carbaryl RS039 Instrument: Dispersive Phase: Potassium Bromide Disc

Carbenoxolone RS041 Instrument: Dispersive Phase: Potassium Bromide Disc

Carbimazole RS042 Instrument: Dispersive Phase : Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Cefalexin RS049 Instrument: Fourier Transform Phase: Potassium Bromide Disc

Cefotaxime Sodium RS044 Instrument: Dispersive Phase: Potassium Bromide Disc

Cefradine RS050 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 400.0

Cefuroxime Axetil RS047 Instrument: Dispersive Phase: Potassium Bromide Disc

2000.0 1800 1600 1400 1200 1000 800 600 400.0

Chlorhexidine RS449 Instrument: Fourier Transform Phase: Potassium Bromide Disc

Chloroform RS053 Instrument: Dispersive Phase: 0.1mm Layer

40 . ¡-------.-.......... + ... ..__... ..._____. . . . . __ 1 ......... _.......•....... -.... + .... -._-.

~ 20 +--·----···· ..·· ·~,, -·~~~-·_·-4H i--·--·-·-·-_·-·+~~~~_·--+-,,- --·- ·-----··r·.. --·-

~ 20 .¡. _ ..•....,. -1- --

40 . ¡-------.-.......... + ... ..... ..._. . . . . 1 ......... _.......•....... -.... + .... -._-.

~ 20 +--·----···· ..·· ·~,, -·~-·_·-4H i--·--·-·-·-_·-·+~~_·--+-,,- --·- ·-----··r·.. --·-