DEVELOPMENTOFUPLCASSAYMETHODFOR

QUETI
API
NEANDI
TSRELATI
VESUBSTANCESSTUDI
ES

THESI
SSUBMI
TTED
TO
BI
JUPATNAI
KUNI
VERSI
TYOFTECHNOLOGY,
ORI
SSA,
ROURKELA
I
npar
ti
alf
ulf
il
lmentoft
her
equi
rement
s
Fort
heawar
doft
hedegr
eeof
MASTEROFPHARMACY
By
Mr
.Pr
aful
laKumarPr
adhan
Regd.No.
:1808254017
Undert
hegui
danceof

Mr
.Sat
ishShar
ma Pr
of.S.K.
Panda
Head,
QC
M.Phar
m,Ph.
D,
I
PCALabor
tor
iesLt
d,
F.
I.
C
Pi
thampur
Pr
inci
pal

RCPHS,
Ber
hampur
 Roy
alCol
legeofPhar
macyandHealthSci
ences
Ber
hampur,Odi
sha,
Indi
a,
2020

Decl
arat
ion

I
,Pr
aful
laKumarPr
adhn,
ast
udentofM.Phar
m.do her
ebydecl
aret
hatt
he
wor
kembodi
edi
nthet
hesi
sent
it
led“
Devel
opmentofUPLCAssaymet
hod
f
orQuet
iapi
neandI
tsRel
ati
veSubst
ancesSt
udi
es”bei
ngsubmi
tt
ed t
othe
Bi
j
uPat
nai
kUni
ver
sit
yofTechnol
ogy
,Rour
kel
a,Odi
shai
npar
ti
alf
ulf
il
lmentof
t
her
equi
rement
sfort
heawar
doft
hedegr
eeofMast
erofPhar
macyi
sthe
bonaf
ide r
esear
ch wor
k car
ri
ed outby me i
nthe l
abor
ator
ies oft
he
Depar
tmentofPhar
maceut
icalAnal
ysi
sandQual
i
tyAssur
ance,Roy
alCol
l
ege
ofPhar
macyandHeal
thSci
ences,
Ber
hampur
,Or
issa,
aff
il
iat
edt
oBi
j
uPat
nai
k
Uni
ver
sit
yofTechnol
ogy
,Odi
sha,Rour
kel
aundert
hesuper
visi
onofPr
of.S.K.
Panda,Pr
inci
paloft
hiscol
l
egeandMr
.Sat
ishShar
ma,Head,QCofI
PCA
Labor
ator
iesLt
d,Pi
thampur
.

If
urt
herdecl
aret
hatt
her
esul
tspr
esent
edi
nthi
sthesi
shav
enotbeen
submi
tt
edel
sewher
efort
heawar
dofanyot
herdegr
eeordi
ploma.

Ber
hampur Pr
aful
laKumarPr
adhan

Dat
e: Regd.No.
:1808254017
 Cer
ti
fi
cat
e

Thi
sist
ocer
ti
fyt
hatt
hewor
kembodi
edi
nthet
hesi
sent
it
led“
Devel
opment
ofUPLCassaymet
hodf
orQuet
iapi
neandI
tsRel
ati
veSubst
ancesSt
udi
es”
bei
ngsubmi
tt
edby
Mr.Pr
aful
laKumarPr
adhant
otheBi
j
uPat
nai
kUni
ver
sit
y
ofTechnol
ogy
,Odi
sha,i
npar
ti
alf
ulf
il
lmentoft
her
equi
rement
sfort
heawar
d
oft
hedegr
eeofMast
erofPhar
macyi
sabonaf
ider
esear
chwor
kcar
ri
edout
byhi
m i
nthel
abor
ator
iesoft
heRoy
alCol
l
egeofPhar
macyandHeal
th
Sci
ences,Ber
hampur
,Depar
tmentofPhar
maceut
icalAnal
ysi
sandQual
i
ty
Assur
ance,undermysuper
visi
on.Ther
esul
tspr
esent
edi
nthi
sthesi
shav
e
notbeensubmi
tt
edel
sewher
efort
heawar
dofanyot
herdegr
ee.

I
n myopi
nion,t
his wor
k has r
eached t
he st
andar
dful
fi
ll
i
ng t
he par
ti
al
r
equi
rement
sfort
he awar
d oft
he degr
ee ofMast
erofPhar
macy i
n
accor
dancewi
tht
her
egul
ati
onsoft
heUni
ver
sit
y.

Pr
of.S.K.Panda
M.Phar
m.,
Ph.D,
F.I
.C
Roy
alCol
legeofPhar
macyand
Heal
thSci
ences,
Ber
hampur
,Odi
sha
Cer
ti
fi
cat
e
 Pr
of.S.K.Panda
M.Phar
m.,
Ph.D.
,F.
I.
C.

Pr
inci
pal
Roy
alCol
legeofPhar
macyand
Heal
thSci
ences,
Ber
hampur

Cer
tif
icat
e
Thi
sist
o cer
ti
fyt
hatt
her
esear
chwor
kdescr
ibedi
nthet
hesi
sent
it
led

“
Dev
elopmentofUPLC assay met
hod f
orQuet
iapi
ne and I
ts Rel
ati
ve

Subst
ancesst
udi
es”bei
ngsubmi
tt
edbyMr
.Pr
aful
l
aKumarPr
adhant
othe

Bi
j
uPat
nai
kUni
ver
sit
yofTechnol
ogy
,Odi
sha,i
npar
ti
alf
ulf
il
lmentoft
he

r
equi
rement
sfort
heawar
doft
hedegr
eeofMast
erofPhar
macyi
scar
ri
ed

outbyhi
mint
hel
abor
ator
iesoft
heDepar
tmentofPhar
maceut
icalAnal
ysi
s

and Qual
i
tyAssur
ance,Roy
alCol
l
egeofPhar
macyand Heal
th Sci
ences,

Ber
hampurundermysuper
visi
onandMr
.Sat
ishShar
ma,Head,QCofI
PCA

Labor
ator
iesLt
d,Pi
thampur
.

Pl
ace: Pr
of.S.K.Panda
Dat
e: Pr
inci
pal
Roy
alCol
legeofPhar
macyand
Heal
thSci
ences,
Ber
hampur
.
 Acknowl
edgement
s

Thesat
isf
act
iont
hatcomeswi
thanysuccessf
ulcompl
eti
onofany
wor
kwoul
dbei
ncompl
eteunl
esswement
iont
henamesofpeopl
ewhomade
i
tpossi
bleandwhoseconst
antgui
danceandencour
agementser
vedasbeam
ofl
i
ghtandcr
ownedoutef
for
ts.
Iexpr
essmydeepsenseofgr
ati
tudet
omygui
dePr
of.S.
K.Pandaf
or
hi
sbenev
olentgui
dance,i
nspi
ri
ng t
hought
sand const
antencour
agement
dur
ing t
he ent
ir
e per
iod ofmyr
esear
ch wor
k.Iacknowl
edge mydeep
i
ndebt
edness t
o hi
m notonl
yforsuch subt
lei
nfl
uences,sci
nti
l
lat
ing
di
scussi
onsbutal
sof
ort
heexampl
eofdedi
cat
ionandl
ovef
orsci
ent
if
ic
r
esear
chhehassetbef
oreme.
It
aket
hisoppor
tuni
tyt
oexpr
essmydeepsenseofgr
ati
tudet
oPr
of.P.
N.Mur
thy
,Di
rect
or,R.
C.P.
S.H,Ber
hampurf
oral
l
owi
ngmet
ocar
ryoutt
his
r
esear
ch wor
k atDepar
tmentofPA&QA,R.
C.P.
H.S Ber
hampurand f
or
pr
ovi
dingal
lnecessar
yrequi
rement
sfort
hiswor
kint
hecol
l
ege.
Iexpr
essmyhear
tfel
tthankst
oPr
of.U.S.Mi
shr
a,Vi
cePr
inci
pal&
HOD,Dept
.ofPA&QA,Pr
of.S.K.SahooandPr
of.R.R.Sar
angif
ort
hei
r
const
anthel
pandv
aluabl
esuggest
iont
ocar
ryoutr
esear
chwor
k.
Iexpr
essmyhear
tfel
tthankst
oDr
.Ar
indom Sen(
PlantHead)
,Mr
.
Sat
ishShar
ma(
Head,QC)
,Mr
.Sant
oshVi
shwakar
ma(
AGM,QC)andMr
.
Pr
akash Pat
idar(
Mananger
,QC)ofI
PCA Labor
ator
ies Lt
d,Pi
thampur
(
Indor
e).
I
twoul
dnothav
ebeenpossi
blet
ocompl
etet
hiswor
kwi
thoutt
he
pr
opi
ti
ouscooper
ati
onandpr
udentcr
it
ici
sm bySur
eshpandasi
r,Babubhai
andAshokSar
karandmanyper
sonsar
oundme.
Lastbutdef
ini
tel
ynott
hel
east
,Ipaymyr
ever
encet
othi
sinst
it
ute,
Roy
alCol
l
egeofPhar
macyandHeal
thSci
ences.Iam undeni
abl
ypr
oudt
obe
associ
atedwi
tht
hiscol
l
ege.

(
Praf
ull
aKumarPr
adhan)
 DEDI
CATEDTO
MYBELOVEDPARENTS,
TEACHERSandI
PCAf
ami
ly
CHAPTER CONTENTS PageNo.

1 I
NTRODUCTI
ON 1-
16

2 DRUGPROFI
LE 17-
18

3 LI
TERATUREREVI
EW 19-
24

RESEARCHENVI
SAGED 25

4 OBJECTI
VESANDPLANOF 26
WORK

5 EXPERI
MENTALWORK 27-
29

6 RESULTSANDDI
SCUSSI
ON 30-
39

7 CONCLUSI
ONS 40

8 REFERENCES 41-
42
 ABBREVATI
ONS
CHEMI
CALS

ACN Acet
oni
tr
il
e

H2O2 Hy
drogenper
oxi
de

Met
h Met
hanol

H2O Wat
er

HCl Hy
drochl
ori
cAci
d

NaOH Sodi
um hy
droxi
de

CONCENTRATI
ONS

L Li
tr
e

M Mol
ari
ty

Mi
n Mi
nut
e

Mm Mi
l
limol
ar

N Nor
mal
i
ty

%W/
W Per
centwei
ghtperwei
ght

%V/
V Per
centv
olumeperv
olume

SYMBOLS

Λ Wav
elengt
h

2
r Cor
rel
ati
oncoef
fi
cient

TEXTABBREVI
ATI
ON
 API Act
ivephar
maceut
ical
ingr
edi
ent

CC Cal
i
brat
ioncur
ve

DAD Di
odear
raydet
ect
or

EI
S/MS/
MS El
ect
ronspr
ayi
oni
zat
ion-massspect
romet
ry

FDA Foodanddr
ugadmi
nist
rat
ion

Fi
g. Fi
gur
e

FT-
IR Four
iert
ransf
ormi
nfr
aredspect
roscopy

GC-
MS Gaschr
omat
ogr
aphy
-massspect
roscopy

HPLC Hi
ghper
for
mancel
i
qui
dchr
omat
ogr
aphy

I
CH I
nter
nat
ional
conf
erenceonhar
moni
zat
ion

I
P I
ndi
anPhar
macopoei
a

LC Li
qui
dchr
omat
ogr
aphy

LOD Li
mitofdet
ect
ion

ODS Oct
adecy
lsi
l
ane

PDA Phot
odi
odear
raydet
ect
or

Rev
ersephase-hi
ghper
for
mancel
i
qui
d
UPLC
chr
omat
ogr
aphy

RSD Rel
ati
vest
andar
ddev
iat
ion

UPLC Ul
tr
aper
for
mancel
i
qui
dchr
omat
ogr
aphy
 TEXTABBREVI
ATI
ON

(
PARAMETERS)

SD St
andar
ddev
iat
ion

Rt Ret
ent
iont
ime

RT Room t
emper
atur
e

UV-
Vis UVv
isi
blespect
roscopy

W.
S Wor
kingst
andar
d

WEI
GHTMEASURES

% Per
cent
age

µ Mi
crons

µg Mi
crogr
am

µl Mi
crol
i
tre

µM Mi
cromet
ers

0
C Degr
eeCel
sius

Conc. Concent
rat
ion

Mg Mi
l
li
gram

Mi
n Mi
nut
es

Ml Mi
l
li
li
tr
e

Mm Mi
l
limet
er

Nm Nanomet
er

Sec Seconds
 LI
STOFTABLES

SL.
TABLE TI
TLE PAGENO.
NO.

1. 6.1 Li
stofI
nst
rument
sUsedDur
ingAnal
ysi
s 28

2. 6.2 Li
stofChemi
cal
sUsedDur
ingAnal
ysi
s 29

3. 8.1 Sol
ubi
l
ityof
Quet
iapi
ne 31

Cal
i
brat
ionTabl
eoft
heUV-
Vis
4. 8.
2 32
Spect
rophot
omet
ri
cMet
hodf
orQuet
iapi
ne

Accur
acyDat
aof
uv-
visi
blespect
roscopy
5. 8.
3 33
met
hodf
orQuet
iapi
ne

Pr
ecessi
onDat
aoft
heUV-
VISspect
roscopy
6. 8.
4 34
met
hodf
orQuet
iapi
ne

I
ntr
aDayPr
ecessi
onDat
aoft
heUV-
VIS
7. 8.
5 34
spect
roscopymet
hodf
orQuet
iapi
ne

I
nter
dayDat
afort
heUV-
VISspect
roscopy
8. 8.
6 34
met
hodf
orQuet
iapi
ne

RuggednessDat
afort
heUV-
VISspect
roscopy
9. 8.
7 35
met
hodf
orQuet
iapi
neanal
i
st(1&2)

RuggednessDat
afort
hedi
ff
erentmodel
ofUV
10. 8.
8 35
-
VISspect
roscopymet
hodf
orQuet
iapi
ne

Robust
nessDat
afort
heUV-
VISspect
roscopy
11 8.
9 met
hodf
orQuet
iapi
nebydi
ff
erent 35
composi
ti
on
 Opt
imi
sedchr
omat
ogr
aphi
ccondi
ti
on
11. 8.
10 36
of
Quet
iapi
nef
orUPLCmet
hod

12. 8.
11 Rel
ati
vesubst
ancei
ndi
l
uent
s 37

Rel
ati
vesubst
ancei
nSy
stem Sui
tabi
l
ity
13. 8.
12 38
Sol
uti
on

14. 8.
13 Rel
ati
vesubst
ancei
nPl
aceboSol
uti
on 39

Rel
ati
vesubst
ancei
nQuet
iapi
neTabl
ets25mg
15. 8.
14 40

LI
STSOFFI
GURES

SL. FI
G. PAGE
NO. NO. NO.
TI
TLE

1. 1.
1 UPLCdi
agr
am 6

2. 2.
1 Quet
iapi
ne 19

3. 8.
1 Ov
erl
ayUV-
VISSpect
raof
Quet
iapi
ne 32

4. 8.
2 Cal
i
brat
ioncur
veof
Quet
iapi
nebyuv
-vi
s 33
spect
rophot
omet
ri
cmet
hod

5. 8.
3 Repr
esent
ati
vechr
omat
ogr
am ofDi
l
uent
s 36

6. 8.
4 Repr
esent
ati
vechr
omat
ogr
am ofSt
andar
dsol
uti
on 37

7. 8.
5 Repr
esent
ati
vechr
omat
ogr
am ofRel
ati
veSubst
ancesi
n 37
di
l
uent
s

8. 8.
6 Repr
esent
ati
vechr
omat
ogr
am of
Rel
ati
veSubst
ancesi
n 38
 Sy
stem Sui
tabi
l
itySol
uti
on

9. 8.
7 Repr
esent
ati
vechr
omat
ogr
am of
Rel
ati
veSubst
ancei
n 39
Pl
acebosol
uti
on

10.8.
8 Repr
esent
ati
veChr
omat
ogr
am ofRel
ati
veSubst
ancei
n 40
Quet
iapi
neTabl
ets25mg

ABSTRACT

A new si
mpl
e, sensi
ti
ve, pr
eci
se and accur
ate ul
tr
a per
for
mance l
i
qui
d
chr
omat
ogr
aphy met
hod has been est
abl
i
shed and v
ali
dat
ed f
oranal
ysi
s of
Quet
iapi
nei
nbul
kandt
abl
etdosagef
orm.

Obj
ect
ive:Todev
elopandv
ali
dat
eUV-
VISSpect
rophot
omet
ryandUl
tr
aper
for
mance
l
i
qui
dchr
omat
ogr
aphi
cmet
hodf
orest
imat
ionofQuet
iapi
nef
rom bul
kandt
abl
et
dosagef
orm.

Met
hod:The spect
rophot
omet
ry met
hod uses met
hanolas a sol
vent and
absor
banceswar
emeasur
ed.Fort
heUPLCmet
hod,
thesepar
ati
onwasachi
evedon
a ACQUI
TY C18 col
umn usi
ng ammoni
um acet
ate:met
hanol(
950:
50) and
acet
oni
tr
il
e:met
hnol
:pur
if
iedwat
er(
550:
400:
50)wi
thaf
lowr
ateof0.
35ml
/mi
nand
det
ect
ionwav
elengt
hat240nm.Themet
hodwasv
ali
dat
edf
orl
i
near
it
y,accur
acy
,
pr
eci
sionandspeci
fi
cit
yasperI
CHgui
del
i
nes.Thedev
elopedandv
ali
dat
edmet
hod
wassuccessf
ull
yusedf
ort
hequant
it
ati
veanal
ysi
sofcommer
cial
l
yav
ail
abl
edosage
f
orm.

Resul
ts:Ther
etent
iont
imeofQuet
iapi
newasf
oundt
obeat4.
137mi
n.Li
near
it
yof
t
he met
hod was f
ound t
o be i
nthe concent
rat
ion r
ange of10-
50 μg/
mlwi
th
cor
rel
ati
oncoef
fi
cientof0.
999.Themet
hodwasv
ali
dat
edf
oraccur
acy
,pr
eci
sion
andr
ecov
eryst
udi
es.

Concl
usi
on:Thehi
ghper
cent
ageofr
ecov
eryandl
owper
cent
ageofr
elat
ivest
andar
d
dev
iat
ionconf
ir
mthesui
tabi
l
ityoft
hemet
hodf
ort
heest
imat
ionofQuet
iapi
nei
n
bul
kandt
abl
etdosagef
orm.

Key
wor
ds:Quet
iapi
ne,
UPLC,
Stabi
l
ityi
ndi
cat
ing
I
NTRODUCTI
ON

Dur
ing l
astf
ew decades,anal
yti
calchemi
str
y has wi
tnessed ext
ensi
ve
dev
elopmenti
nter
msofsophi
sti
cat
ion,quant
it
ati
onandi
nst
rument
ati
on.
Consequent
lyneweranal
yti
calt
echni
ques(
suchashy
phenat
edt
echni
ques
l
i
keUPLC,GC,LC-
MS,GC-
MS et
c.)and t
hei
rar
easofappl
i
cat
ionshav
e
i
ncr
eased consi
der
abl
y because of st
ri
ngent r
equi
rement f
or t
est
ing
moni
tor
ingoft
hedr
ugsf
orappr
oval
,thedemandonqual
i
tyv
ali
dat
iondat
a
andper
for
manceofanal
yti
cal
met
hodshav
egai
nedani
mpor
tance.1,
2

Oneoft
hemostdi
ff
icul
tsi
tuat
ioni
sthataphar
maceut
icalanal
ystmay
hav
e conf
ront
ed wi
tht
he sel
ect
ion ofsui
tabl
e anal
yti
calmet
hod f
ora
par
ti
cul
arpr
obl
em.Ther
efor
e,t
her
ear
esev
eralv
ali
dreasonsf
ordev
elopi
ng
new met
hods. Di
ff
erent t
ypes of anal
yti
cal t
echni
ques l
i
ke
2
spect
rophot
omet
ri
c,chr
omat
ogr
aphi
c,et
c.ar
eav
ail
abl
e.

1.
1METHODDEVELOPMENT

1.
1. CMETHOD3,4
1SPECTROPHOTOMETRI

Spect
rophot
omet
ri
cgener
all
ybasedonmeasur
ingt
heabsor
pti
onofa
monochr
omat
icl
i
ghtbycol
our
lesscompoundsnearUV pat
hofspect
rum
(
200-
400nm)
.Thephot
omet
ri
cmet
hodofanal
ysi
sisbasedont
heLamber
t-
Beer
’sl
aw.Thi
slaw suggestt
hatt
heabsor
banceofasol
uti
oni
sdi
rect
ly
pr
opor
ti
onal
tot
heconcent
rat
ionofsol
ute(
anal
yte)
.

Thei
mpor
tantappl
i
cat
ionsar
e,

 I
dent
if
icat
ionofmanyt
ypesofor
gani
c,i
nor
gani
cmol
ecul
esandi
ons.
.

 Quant
it
ati
vedet
ermi
nat
ionofmi
xtur
esofanal
ytes.

 Moni
tor
ingandi
dent
if
icat
ionofchr
omat
ogr
aphi
cofef
fl
uent
s.

 Det
ermi
nat
ionofequi
l
ibr
ium const
ant
s.

 Moni
tor
ingr
ateofr
eact
ion.

1,
2,
3,
5
1.
1.2ULTRA PERFORMANCELI
QUI
DCHROMATOGRAPHY(
UPLC)

R.
C.P.
H.S,
BERHAMPURPage1
 The t
erm chr
omat
ogr
aphy def
ines t
he separ
ati
on oft
he v
ari
ous
speci
esofami
xtur
eont
hebasi
soft
hei
rdi
str
ibut
ionchar
act
eri
sti
csbet
ween
ast
ati
onar
yandamobi
l
ephase.UPLC t
echni
quei
suset
oov
ercomet
he
l
i
mit
ati
onsofcol
umnchr
omat
ogr
aphysuchassl
owsepar
ati
onandpoorf
low
r
ate.Thesemet
hodsar
ehi
ghl
yaut
omat
edandext
remel
ysensi
ti
ve.UPLC
cont
ainpr
essur
epumpf
ori
ncr
easef
lowr
ateofsol
vent
,ef
fi
cientsepar
ati
on.
Col
umnsar
epackedwi
thsmal
lpar
ti
clest
hatpr
ovi
debet
termasst
ransf
er.
Var
iet
iesofon-
li
nedet
ect
orar
efl
exi
blet
ovar
iet
yofappl
i
cat
ion.

Pr
inci
pleofUPLC

Thepr
inci
pleofsepar
ati
oni
sadsor
pti
on.Separ
ati
onofcomponent
s
t
akespl
acebecauseoft
hedi
ff
erencei
naf
fi
nit
yofcomponent
stowar
ds
st
ati
onar
yphase.Thepr
inci
plei
sseeni
nnor
malphaseaswel
lasr
ever
se
phasewher
eadsor
pti
ont
akespl
ace.

Met
hods

Themoder
nfor
m ofcol
umnchr
omat
ogr
aphyhasbeencal
l
edhi
gh
per
for
mance, hi
gh pr
essur
e, hi
gh r
esol
uti
on and hi
gh speed l
i
qui
d
chr
omat
ogr
aphy
.

Ul
tr
aPer
for
manceLi
qui
dChr
omat
ogr
aphy(
UPLC)i
saspeci
albr
anch
ofcol
umnchr
omat
ogr
aphyi
nwhi
cht
hemobi
l
ephasei
sfor
cedt
hrought
he
col
umnathi
ghspeed.Asar
esul
ttheanal
ysi
sti
mei
sreducedby1-
2or
der
sof
magni
tuder
elat
ivet
ocl
assi
calcol
umnchr
omat
ogr
aphyandt
heuseofmuch
smal
l
erpar
ti
clesoft
headsor
bentorsuppor
tincr
easest
hecol
umnef
fi
ciency
.

The essent
ialequi
pmentconsi
sts ofan el
uent
,reser
voi
r,a hi
gh
pr
essur
epump,f
orani
nject
ori
ntr
oduci
ngt
hesampl
e,acol
umncont
aini
ng
t
hest
ati
onar
yphase,adet
ect
orandar
ecor
der
.Thedev
elopmentofhi
ghl
y
ef
fi
cientmi
cropar
ti
cul
atebondedphaseshasi
ncr
easedt
hev
ersat
il
it
yoft
he
t
echni
queandhasgr
eat
lyi
mpr
ovedt
heanal
ysi
sofmul
ticomponentmi
xtur
es.

Thev
ari
ouscomponent
sofaUPLCsy
stem ar
edescr
ibedbel
ow,

Sol
ventcont
ainer Pump Dampi
nguni
t Sampl
einj
ect
or Col
umn

R.
C.P.
H.S,
BERHAMPURPage2
 Det
ect
or

1.Sol
ventReser
voi
r:
Mobi
l
ephasecont
ent
sar
econt
ainedi
nagl
assr
eser
voi
r.Themobi
l
e
phase,orsol
vent
,inUPLCi
susual
l
yami
xtur
eofpol
arandnon-
pol
arl
i
qui
d
component
swhoser
espect
iveconcent
rat
ionsar
evar
ieddependi
ngont
he
composi
ti
onoft
hesampl
e.Reser
voi
rmadeupofst
ainl
essst
eelandcapaci
ty
about1000ml
.UPLC i
nst
rumental
so cont
ains degassersy
stem;Mai
n
f
unct
ionofdegassersy
stem i
stor
emov
efi
nebubbl
esofi
ner
tgas.Degasser
sy
stem cont
ainsv
acuum pumpi
ngsy
stem,di
sti
l
lat
ionsy
stem,heat
ingand
st
ir
ri
ngofsol
vent
s.
2.Pump:
A pumpaspi
rat
est
hemobi
l
ephasef
rom t
hesol
ventr
eser
voi
rand
f
orcesi
tthr
ought
hesy
stem’
scol
umnanddet
ect
or.Dependi
ngonanumber
off
act
orsi
ncl
udi
ngcol
umndi
mensi
ons,par
ti
clesi
zeoft
hest
ati
onar
yphase,
t
hef
lowr
ateandcomposi
ti
onoft
hemobi
l
ephase,
oper
ati
ngpr
essur
esofup
t
o10000-
12000psicanbegener
ated.Tor
educev
ari
ati
oni
ntheel
uti
on,t
he
pumpmustmai
ntai
naconst
ant
,pul
sef
ree,f
low r
ate;t
hisi
sachi
evedwi
th
mul
ti
-pi
stonpumps.Thepr
esenceoft
wopi
stonsal
l
owst
hef
low r
atet
obe
cont
rol
l
edbyonepi
stonast
heot
herr
echar
ges.Asy
ringepumpcanbeused
f
orev
engr
eat
ercont
roloff
lowr
ate;howev
er,t
hesy
ringepumpi
sunabl
eto
pr
oduceasmuchpr
essur
easapi
stonpump,
soi
tcannotbeusedi
nal
lUPLC
appl
i
cat
ions.
3.Sampl
eInj
ect
or:
Thei
nject
orcanbeasi
ngl
einj
ect
ionoranaut
omat
edi
nject
ionsy
stem.
Ani
nject
orf
oranUPLCsy
stem shoul
dpr
ovi
dei
nject
ionoft
hel
i
qui
dsampl
e
wi
thi
nther
angeof0.
1-100mLofv
olumewi
thhi
ghr
epr
oduci
bil
i
tyandunder
hi
ghpr
essur
e(upt
o12000psi
).

Themostpopul
aronei
sRheody
nei
nject
or.Thi
shasaf
ixedv
olume
l
oopl
i
ke20or50ormor
e.I
thast
womodesi
.el
oadposi
ti
onwhent
hesampl
e
i
sloadedi
nthel
oopandt
hei
njectmodewhent
hesampl
eisi
nject
ed.

R.
C.P.
H.S,
BERHAMPURPage3
4.Col
umns:
Col
umns ar
e usual
l
y made of pol
i
shed st
ainl
ess st
eel
,gl
ass,
pol
yet
hyl
eneandAcqui
tycol
umnar
ebet
ween5cm t
o30cm l
ongandhav
e
ani
nter
naldi
amet
erofbet
ween2and50mm.Theyar
ecommonl
yfi
l
ledwi
th
ast
ati
onar
yphasewi
thapar
ti
clesi
zeof3–10µm.Col
umnswi
thi
nter
nal
di
amet
ersofl
esst
han2mm ar
eof
tenr
efer
redt
oasmi
crobor
ecol
umns.
I
deal
l
ythet
emper
atur
eoft
hemobi
l
ephaseandt
hecol
umnshoul
dbekept
const
antdur
ingananal
ysi
s.
The f
unct
ionalgr
oup pr
esenti
nthe st
ati
onar
y phase ofcol
umn
dependsont
het
ypeofsepar
ati
on.

Tabl
e1.
1:Ty
pesoff
unct
ionalgr
ouppr
esenti
nthecol
umn

PHASES FUNCTI
ONALGROUP MODE OR TYPE SEPARATI
ONEFFI
CIENCY
OFSEPARATI
ON

Si Si
l
ica Nor
mal
phase Forpol
arnoni
oni
cor
gani
c
compounds.

C1 Tr
imet
hyl
sil
ane(
TMS) Rev
ersedphase Forpol
arandmul
ti
funct
ional
compounds.

C3 pr
opy
l Rev
ersedphase Forpr
otei
nsandpept
ides

C4 But
yl Rev
ersedphase For l
arge pr
otei
ns and
hy
drophobi
cpept
ides.

C6 Hexy
l Rev
ersedphase For i
on pai
ri
ng
chr
omat
ogr
aphy
.

C8 Oct
yl,
RP-8,
LC8 Rev
ersedphase For pept
ides, st
eroi
ds,
nucl
eosi
des,
phar
maceut
ical
s and smal
l
hy
drophi
l
icpr
otei
ns.

C18 ODS, RP-

18, LC18, For non-
pol
ar sol
utes,

R.
C.P.
H.S,
BERHAMPURPage4
 Oct
adecy
l pept
ides, st
eroi
ds, f
att
y
aci
ds,
nucl
eosi
des,

5.Det
ect
or
TheUPLC det
ect
or,l
ocat
ed att
heend oft
hecol
umndet
ect
sthe
anal
ytesast
heyel
utef
rom t
hechr
omat
ogr
aphi
ccol
umnandi
tdependson
t
hepr
oper
tyoft
hecompoundst
obesepar
ated.Commonl
yuseddet
ect
ors
ar
e UV-det
ect
or,r
efr
act
ivei
ndex,f
luor
escence,mass-
spect
romet
ri
c and
el
ect
rochemi
cal
det
ect
ors.
Ther
ecentonei
sphot
odi
odear
raydet
ect
or(
PDA det
ect
or)whi
chi
s
si
mil
art
oUVdet
ect
orwhi
choper
atesbet
ween190-600nm.Ther
esul
ti
ng
spect
rum i
s 3-
D ort
hree di
mensi
onalpl
otofResponse Vs Ti
me Vs
Wav
elengt
h.
6.Recor
der
s:
Si
gnal
sfr
om t
hedet
ect
ormaybecol
l
ect
ed on char
trecor
der
sor
el
ect
roni
cint
egr
ator
sthatv
aryi
ncompl
exi
tyandi
nthei
rabi
l
ityt
opr
ocess,
st
oreand r
epr
ocesschr
omat
ogr
aphi
cdat
a.The comput
eri
ntegr
atest
he
r
esponse of t
he det
ect
or t
o each component and pl
aces i
tint
oa
chr
omat
ogr
apht
hati
seasyt
oreadandi
nter
pret
.
7.Chr
omat
ogr
am

A chr
omat
ogr
am i
sar
epr
esent
ati
on oft
he separ
ati
on t
hathas
chemi
cal
l
y[chr
omat
ogr
aphi
cal
l
y]occur
redi
ntheUPLCsy
stem.Aser
iesof
peaksr
isi
ngf
rom abasel
i
nei
sdr
awnonat
imeaxi
s.Eachpeakr
epr
esent
s
t
hedet
ect
orr
esponsef
oradi
ff
erentcompound.Thechr
omat
ogr
am i
spl
ott
ed
byt
hecomput
erdat
ast
ati
on.

The out
putwi
l
lbe r
ecor
ded as a ser
ies ofpeaks -each one
r
epr
esent
ingacompoundi
nthemi
xtur
epassi
ngt
hrought
hedet
ect
orand
absor
bingUVl
i
ght
.Asl
ongasy
ouwer
ecar
efult
ocont
rolt
hecondi
ti
onson
t
he col
umn,y
ou coul
d use t
he r
etent
ion t
imes t
o hel
ptoi
dent
if
ythe
compoundspr
esent-pr
ovi
dedt
hathadal
readymeasur
edt
hem f
orpur
e
sampl
esoft
hev
ari
ouscompoundsundert
hosei
dent
ical
condi
ti
ons.

R.
C.P.
H.S,
BERHAMPURPage5
1.
1. 1METHODDEVELOPMENT8,
2. 9

Met
hod f
oranal
yzi
ng dr
ug can be dev
eloped,pr
ovi
ded one has
knowl
edgeaboutt
henat
ureoft
hesampl
e,namel
y,i
tsmol
ecul
arwei
ght
,
pol
ari
ty,
ioni
cchar
act
erandt
hesol
ubi
l
itypar
amet
er.Anexactr
eci
pef
orUPLC,
howev
er,cannot be pr
ovi
ded because met
hod dev
elopment i
nvol
ves
consi
der
abl
etr
ialander
rorpr
ocedur
es.Themostdi
ff
icul
tpr
obl
em usual
l
yis
wher
etost
art
,whatt
ypeofcol
umni
swor
tht
ryi
ngwi
thwhatki
ndofmobi
l
e
phasei
ngener
al,onebegi
nswi
thr
ever
sedphasechr
omat
ogr
aphy
,whent
he
compoundsar
ehy
drophi
l
ici
nnat
urewi
thmanypol
argr
oupsandar
ewat
er
sol
ubl
e.

El
uti
onofdr
ugmol
ecul
escanbeal
ter
edbychangi
ngt
hepol
ari
tyoft
he
mobi
l
ephase.Theel
uti
onst
rengt
hofamobi
l
ephasedependsuponi
ts
pol
ari
ty,
thest
rongert
hepol
ari
ty,
higheri
stheel
uti
on.I
oni
csampl
es(
aci
dicor
basi
c) can be separ
ated,i
fthey ar
e pr
esent i
n undi
ssoci
ated f
orm.
Di
ssoci
ati
onofi
oni
csampl
esmaybesuppr
essedbypr
opersel
ect
ionofpH.

ThepHoft
hemobi
l
ephasehast
obesel
ect
edi
nsuchawayt
hatt
he
compoundsar
enoti
oni
zed.I
fther
etent
iont
imesar
etooshor
t,t
hedecr
ease
oft
heor
gani
cphaseconcent
rat
ioni
nthemobi
l
ephasecanbei
nst
epsof5%.
I
fther
etent
iont
imear
etool
ong,
ani
ncr
easei
n5%st
epsoft
heor
gani
cphase
concent
rat
ioni
sneeded.

Opt
imi
zat
ioncanbest
art
edonl
yaf
terar
easonabl
echr
omat
ogr
am has
beenobt
ained.Ar
easonabl
echr
omat
ogr
am meanst
hatal
lthecompounds
ar
edet
ect
edbymor
eorl
esssy
mmet
ri
calpeaksont
hechr
omat
ogr
am.Bya
sl
i
ghtchangeoft
hemobi
l
ephagecomposi
ti
on,t
heshi
ft
ingoft
hepeakscan
beex
pect
ed.Fr
om af
ew exper
iment
almeasur
ement
s,t
heposi
ti
onoft
he
peaks can be pr
edi
cted wi
thi
nthe r
ange ofi
nvest
igat
ed changes.An
opt
imi
zedchr
omat
ogr
am i
stheonei
nwhi
chal
lthepeaksar
esy
mmet
ri
cal
andar
ewel
lsepar
atedi
nlessr
unt
ime.

Thepeakr
esol
uti
oncanbei
ncr
easedbyusi
ngamor
eef
fi
cientcol
umn
(
col
umnwi
thhi
ghert
heor
eti
calpl
atenumber
,N)
,whi
chcanbeachi
evedby
usi
ngacol
umnofsmal
l
erpar
ti
clesi
ze,oral
ongercol
umn.Thesef
act
ors,

R.
C.P.
H.S,
BERHAMPURPage6
howev
er,wi
l
lincr
ease t
he anal
ysi
sti
me.Fl
ow r
ate does noti
nfl
uence
r
esol
uti
on,
buti
thasast
rongef
fectont
heanal
ysi
sti
me.

St
epsf
orUPLCmet
hoddev
elopment
,
I
nfor
mat
iononsampl
e

Def
ini
ngsepar
ati
ongoal
s

Speci
alpr
ocedur
erequi
rement
,sampl
epr
etr
eat
ment
,ifany

Det
ect
orsel
ect
ionandset
ti
ng

Separ
ati
oncondi
ti
onsopt
imi
zat
ion

Checki
ngf
orpr
obl
emsorspeci
alpr
ocedur
erequi
rement
s

Recov
eryofpur
if
iedmat
eri
al

Quant
it
ati
vecal
i
brat
ion/Qual
i
tat
ivemet
hod

Met
hodv
ali
dat
ionf
orr
eleaset
olabor
ator
ies

1.
1.2.
2METHODOPTI
MI ON8,
ZATI 9

Dur
ingt
heopt
imi
zat
ionst
age,t
hei
nit
ialset
sofcondi
ti
onst
hathav
e
ev
olv
edf
rom t
hef
ir
stst
agesofdev
elopmentar
eimpr
ovedormaxi
mizedi
n
t
ermsofr
esol
uti
onandpeakshape,pl
atecountasy
mmet
ry,capaci
ty,el
uti
on
t
ime,det
ect
ionl
i
mit
s,l
i
mitofquant
it
ati
onandov
eral
labi
l
ityt
oquant
if
ythe
speci
fi
canal
yteofi
nter
est
.

Opt
imi
zat
ionofamet
hodcanf
oll
owei
theroft
wogener
alappr
oaches:

1.Manual

2.Comput
erdr
iven

R.
C.P.
H.S,
BERHAMPURPage7
 Themanualappr
oachesi
nvol
vev
ary
ingoneexper
iment
alv
ari
abl
eata
t
ime,Whi
l
ehol
dingal
lot
herconst
ant
sandr
ecor
dingchangesi
nresponse.
Thev
ari
abl
esmi
ghti
ncl
udef
loer
ate,
mobi
l
eorst
ati
onar
yphasecomposi
ti
on,
t
emper
atur
e,det
ect
ion wav
elengt
h.And pH t
his uni
var
iat
e appr
oach t
o
sy
stem opt
imi
zat
ion i
s sl
ow,t
ime consumi
ng and pot
ent
ial
l
yexpensi
ve.
Howev
er,i
tmaypr
ovi
deamuchbet
terunder
standi
ngoft
hepr
inci
plesand
t
heor
yinv
olv
edandofi
nter
act
ionsoft
hev
ari
abl
es.

I
n t
he second appr
oach, comput
er dr
iven aut
omat
ed met
hods
dev
elopment
,ef
fi
ciencyi
sopt
imi
zedwhi
l
eexper
iment
ali
nputi
smi
nimi
zed.
Comput
erdr
ivenaut
omat
edappr
oachescanbeappl
i
edt
omanyappl
i
cat
ions.
I
naddi
ti
on,t
heyar
ecapabl
eofsi
gni
fi
cant
lyr
educi
ngt
het
ime,ener
gyand
costofv
irt
ual
l
yal
l
-inst
rument
almet
hodsdev
elopment
.

The v
ari
ous par
amet
erst
hat i
ncl
ude t
o be opt
imi
zed dur
ing met
hod
dev
elopment

1.Modeofsepar
ati
on

2.Sel
ect
ionofst
ati
onar
yphase

3.Sel
ect
ionofmobi
l
ephase

4.Sel
ect
ionofdet
ect
or

Sel
ect
ionofModeofsepar
ati
on

I
nrev
ersephasemode,t
hemobi
l
ephasei
scompar
ati
vel
ymor
epol
ar
t
hant
hest
ati
onar
yphase.Fort
hesepar
ati
onofpol
arormoder
atel
ypol
ar
compounds,
themostpr
eff
eredmodei
srev
ersephase.Thenat
ureofanal
yte
i
sthepr
imar
yfact
ori
nthesel
ect
ionoft
hemodeofsepar
ati
on.Asecond
f
act
ori
sthenat
ureoft
hemat
ri
x.

Sel
ect
ionofst
ati
onar
yphase/col
umn

Sel
ect
ionoft
hecol
umni
sthef
ir
standmosti
mpor
tantst
epi
nmet
hod

R.
C.P.
H.S,
BERHAMPURPage8
dev
elopment
.Theappr
opr
iat
echoi
ceofsepar
ati
oncol
umni
ncl
udest
hree
di
ff
erentappr
oaches.

1.Sel
ect
ionofsepar
ati
onsy
stem

2.Thepar
ti
clesi
zeandt
henat
ureoft
hecol
umnpacki
ng

3.Thephy
sical
par
amet
eroft
hecol
umni
.e.t
hel
engt
handt
hedi
amet
er

Some of t
he i
mpor
tant par
amet
ers consi
der
ed whi
l
e sel
ect
ing
chr
omat
ogr
aphi
ccol
umnsar
e

 l
engt
handt
hedi
amet
erofcol
umn

 Packi
ngmat
eri
al

 Shapeofpar
ti
cles

 Si
zeofpar
ti
cles

 %ofcar
bonl
oadi
ng

 Por
evol
ume

 Sur
facear
ea

 Endcappi
ng

Thecol
umni
ssel
ect
eddependi
ngont
henat
ureoft
hesol
uteandt
he
i
nfor
mat
ion aboutanal
yte.Rev
ersed phasemodeoft
hechr
omat
ogr
aphy
f
aci
l
itat
esawi
der
angeofcol
umnl
i
kedi
met
hylsi
l
ane(
C2)
,but
ylsi
l
ane(
C4)
,
oct
ylsi
l
ane(
C8)
,acqul
tyCSH(
C18)
,basedeact
ivat
edsi
l
ane(
C18)BDSpheny
l,
cy
anopr
opy
l(CN)
,ni
tr
o,ami
noet
c.C18 waschosenf
ort
hisst
udysi
ncei
tis
mostr
etent
iveone.Thesampl
emani
pul
ati
onbecomeseasi
erwi
tht
hist
ype
ofcol
umn.

Gener
all
ylongercol
umn pr
ovi
des bet
tersepar
ati
on due t
o hi
gh
t
heor
eti
cal
plat
enumber
s.Ast
hepar
ti
clesi
zedecr
eases t
he sur
face ar
e
av
ail
abl
eforcoat
ingi
ncr
ease.Col
umnwi
th5-
µm par
ti
clesi
zegi
vet
hebest
compr
omi
se ofef
fi
ciency
,repr
oduci
bil
i
tyand r
eli
abi
l
ity
.Int
hiscase,t
he

R.
C.P.
H.S,
BERHAMPURPage9
col
umnsel
ect
edhadapar
ti
clesi
zeof5µm andi
nter
nal
diamet
erof4.
6mm.

Peakshapei
sequal
l
yimpor
tanti
nmet
hoddev
elopment
.Col
umnst
hat
pr
ovi
de sy
mmet
ri
calpeaks ar
e al
way
s pr
efer
red whi
l
e peaks wi
th poor
asy
mmet
rycanr
esul
tin.

 I
naccur
atepl
atenumberandr
esol
uti
onmeasur
ement

 I
mpr
eci
sequant
it
ati
on

 Degr
adeandundet
ect
edmi
norbandsi
nthepeakt
ail

 Poorr
etent
ionr
epr
oduci
bil
i
ty

Ausef
ulandpr
act
icalmeasur
ementofpeakshapei
speakasy
mmet
ry
f
act
orandpeakt
ail
i
ngf
act
or.Peakasy
mmet
ryi
smeasur
edat10% off
ull
peakhei
ghtandpeakt
ail
i
ngf
act
orat5%|
.Repr
oduci
bil
i
tyofr
etent
iont
imes
andcapaci
tyf
act
ori
simpor
tantf
ordev
elopi
ng ar
ugged and r
epeat
abl
e
met
hod.

Acol
umnwhi
chgi
vessepar
ati
onofal
lthei
mpur
it
iesanddegr
adant
s
f
rom eachot
herandf
rom anal
ytepeakandwhi
chi
sruggedf
orv
ari
ati
oni
n
mobi
l
ephaseshal
lbesel
ect
ed.

Sel
ect
ionofmobi
lephase

Thepr
imar
yobj
ect
ivei
nsel
ect
ionandopt
imi
zat
ionofmobi
l
ephasei
s
t
oachi
eveopt
imum separ
ati
onofal
lthei
ndi
vi
duali
mpur
it
iesanddegr
adant
s
f
rom eachot
herandf
rom anal
ytepeak.

I
nli
qui
dchr
omat
ogr
aphy
,thesol
uter
etent
ioni
sgov
ernedbyt
hesol
ute
di
str
ibut
ionf
act
or,whi
chr
efl
ect
sthedi
ff
erenti
nter
act
ionsoft
hesol
ute–
st
ati
onar
yphase,sol
ute–mobi
l
ephaseandt
hemobi
l
ephase–st
ati
onar
y
phase.Foragi
venst
ati
onar
yphase,
ther
etent
ionoft
hegi
vensol
utedepends
di
rect
lyupont
hemobi
l
ephase,
thenat
ureandcomposi
ti
onofwhi
chhast
obe
adapt
edi
nter
msofel
uti
onst
rengt
h(sol
uter
etent
ion)andsol
ventsel
ect
ivi
ty
(
sol
ute separ
ati
on)sol
ventpol
ari
tyi
sthe keywor
din chr
omat
ogr
aphi
c
separ
ati
onssi
nceapol
armobi
l
ephasewi
l
lgi
ver
iset
olowsol
uter
etent
ioni
n

R.
C.P.
H.S,
BERHAMPURPage10
nor
malphaseandhi
ghsol
uter
etent
ioni
nrev
ersephaseLC.Thesel
ect
ivi
ty
wi
l
lbepar
ti
cul
arl
yal
ter
edi
fthebuf
ferpHi
scl
oset
othepKaoft
heanal
ytes;
t
hesol
ventst
rengt
hisameasur
eofi
tsabi
l
ityt
opul
lanal
ytef
rom t
hecol
umn.
I
tisgener
all
ycont
rol
l
edbyt
heconcent
rat
ionoft
hesol
ventwi
tht
hehi
ghest
st
rengt
h.

The f
oll
owi
ng ar
ethe par
amet
ers,whi
ch shal
l be t
aken i
nto
consi
der
ati
onwhi
l
esel
ect
ingandopt
imi
zi
ngt
hemobi
l
ephage.

a.Buf
fer

b.pHoft
hebuf
fer

c.mobi
l
ephasecomposi
ti
on

a.Buf
fer
,ifanyandi
tsl
engt
h

Buf
ferandi
tsst
rengt
hpl
ayani
mpor
tantr
olei
ndeci
dingt
hepeak
sy
mmet
ri
esandsepar
ati
ons.Someoft
hemost
,commonl
yempl
oyedbuf
fer
s
ar
e

 Phosphat
e buf
fer
s pr
epar
ed usi
ng sal
tsl
i
ke KH2PO4,K2HPO4,
NaH2PO4,
Na2HPO4et
c

 Phosphor
icaci
dbuf
fer
spr
epar
edusi
ngH3PO4

 Acet
atebuf
fer
s–ammoni
um acet
ate,
sodi
um acet
ateet
c

 Acet
icaci
dbuf
fer
spr
epar
edusi
ngCH3COOH.

Ther
etent
iont
imesal
sodependont
hemol
arst
rengt
hsoft
hebuf
fer–
Mol
arst
rengt
hisi
ncr
easi
ngl
ypr
opor
ti
onalt
oret
ent
iont
imes.Thest
rengt
hof
t
hebuf
fercanbei
ncr
eased,
ifnecessar
y,t
oachi
evet
her
equi
redsepar
ati
on.

Thesol
ventst
rengt
hisameasur
eofi
tsabi
l
ityt
opul
lanal
ytesf
rom t
he
col
umn.I
tisgener
all
ycont
rol
l
edbyt
heconcent
rat
ionoft
hesol
ventwi
tht
he
hi
ghestst
rengt
h.

b.pHoft
hebuf
fer
R.
C.P.
H.S,
BERHAMPURPage11
 pH pl
ays an i
mpor
tant r
olei
n achi
evi
ng t
he chr
omat
ogr
aphi
c
separ
ati
onsasi
tcont
rol
stheel
uti
onpr
oper
ti
esbycont
rol
l
ingt
hei
oni
zat
ion
char
act
eri
sti
cs.Exper
iment
swer
econduct
edusi
ngbuf
fer
shav
ingdi
ff
erent
pHt
oobt
aint
her
equi
redsepar
ati
ons.

I
tisi
mpor
tantt
omai
ntai
nthepHoft
hemobi
l
ephasei
nther
angeof
2.
0to8.
0asmostcol
umnsdoesnotwi
thst
andt
othepHwhi
char
eout
side
t
hisr
ange.Thi
sisduet
othef
actt
hatt
hesi
l
oxanel
i
nkagear
eacl
eav
edbel
ow
pH2.
0,whi
l
epHv
aluedabov
e8.
0si
l
icamaydi
ssol
ve.

c.mobi
lephasecomposi
ti
on

Mostchr
omat
ogr
aphi
csepar
ati
onscanbeachi
evedbychoosi
ngt
he
opt
imum mobi
l
ephasecomposi
ti
on.Thi
sisduet
othef
actt
hatf
air
lyl
arge
amountofsel
ect
ivi
ty can be achi
eved by choosi
ng t
he qual
i
tat
ive and
quant
it
ati
vecomposi
ti
onofaqueousandor
gani
cpor
ti
ons.Mostwi
del
yused
sol
vent
sinr
ever
sephasechr
omat
ogr
aphyar
emet
hanoland acet
oni
tr
il
e.
Exper
iment
swer
econduct
edwi
thmobi
l
ephaseshav
ingbuf
fer
swi
thdi
ff
erent
pHanddi
ff
erentor
gani
cphasest
ocheckf
ort
hebestsepar
ati
onsbet
ween
t
hei
mpur
it
ies.Amobi
l
ephasewhi
chgi
vessepar
ati
onofal
lthei
mpur
it
iesand
degr
adant
sfr
om eachot
herandf
rom anal
ytepeakandwhi
chi
sruggedf
or
v
ari
ati
onofbot
haqueousandor
gani
cphasebyat
least+_0.
2% oft
he
sel
ect
edmobi
l
ephasecomposi
ti
on.

Sel
ect
ionofdet
ect
or

Thedet
ect
orwaschosendependi
nguponsomechar
act
eri
sti
cpr
oper
ty
oft
he anal
ytel
i
ke UV absor
bance,f
luor
escence,conduct
ance,oxi
dat
ion,
r
educt
ionet
c.char
act
eri
sti
cst
hatar
eful
fi
ll
edbyadet
ect
ort
ousedi
nUPLC
det
ermi
nat
ionar
e,

 Hi
ghsensi
ti
vi
ty,
faci
l
itat
ingt
raceanal
ysi
s

 Negl
i
gibl
ebasel
i
nenoi
se.Tof
aci
l
itat
elowerdet
ect
ion

 Lar
gel
i
neardy
nami
crange

 Lowdeadv
olume

R.
C.P.
H.S,
BERHAMPURPage12
  Nondest
ruct
ivet
osampl
e

 I
nexpensi
vet
opur
chaseandoper
ate

Phar
maceut
icali
ngr
edi
ent
sdonotal
labsor
bUVl
i
ghtequal
l
y,sot
hat
sel
ect
ionofdet
ect
ionwav
elengt
hisi
mpor
tant
.Anunder
standi
ngoft
heUV
l
i
ght absor
pti
ve pr
oper
ti
es of t
he or
gani
cimpur
it
ies and t
he act
ive
phar
maceut
ical
ingr
edi
enti
sver
yhel
pful
.

Fort
hegr
eat
estsensi
ti
vi
tyλmaxshoul
dbeused.UV wav
elengt
hs
bel
ow 200nm shoul
dbeav
oidedbecausedet
ect
ornoi
sei
ncr
easesi
nthi
s
r
egi
on.Hi
gherwav
elengt
hsgi
vegr
eat
ersel
ect
ivi
ty.

1.
2GUI
DELI
NESOFRELATI
VEI
MPURI
TIESSTUDTI
ES

The I
CH gui
del
i
nes on “
Rel
ati
veI
mpur
it
y Test
ing on New Dr
ug
Subst
ancesandPr
oduct
s”st
atet
hef
oll
owi
ngf
ordi
ff
erentr
elat
ivei
mpur
it
ies,
“
Iti
srecogni
zedt
hatsomeRel
ati
veI
mpur
it
iesar
eint
erf
erei
ntheanal
ysi
sof
cer
tai
nact
ivephar
maceut
ical
ingr
edi
ent
s”.

Rel
ati
veI
mpur
it
iesst
udyi
sthepr
ocessofsubj
ect
ingdr
ugcompound
t
odi
ff
erentchr
omat
ogr
aphycondi
ti
ont
ofi
ndoutwhi
chi
mpur
it
iesi
nter
fer
e
dur
inganal
ysi
sandhowt
oov
ercomet
hati
nter
fer
ence.

Thesest
udi
esal
sopr
ovi
dei
nfor
mat
ionaboutt
her
elat
ivei
mpur
it
ies
pat
hway dur
ing anal
ysi
s ofdr
ugs.Rel
ati
veI
mpur
it
ies st
udi
es may hel
p
f
aci
l
itat
e phar
maceut
ical dev
elopment i
n ar
eas such as f
ormul
ati
on
dev
elopment
,manuf
act
uri
ngandpacki
ng.

Obj
ect
ivesofRel
ati
veI
mpur
it
iesst es52
udi

Test
ingofsampl
esi
srequi
redt
odemonst
rat
ethef
oll
owi
ngabi
l
iti
esof
anal
yti
cal
techni
queempl
oyedi
nrel
ati
vei
mpur
it
iesst
udi
es

 Todet
ermi
nest
ruct
uralt
ransf
ormat
ionsoft
hedr
ugsubst
ance
anddr
ugpr
oduct
.

 Todet
ectl
owconcent
rat
ionofr
elat
ivei
mpur
it
ies.

R.
C.P.
H.S,
BERHAMPURPage13
  To det
ectunr
elat
ed i
mpur
it
ies i
n pr
esence oft
he desi
red
pr
oductandpr
oductr
elat
eddegr
adant
s.

 Tosepar
atepr
oductr
elat
eddegr
adant
sfr
om t
hoseder
ivedf
rom
exci
pient
sandi
ntactpl
acebo.

 Toel
uci
dat
epossi
bler
elat
ivei
mpur
it
iesst
udypat
h-way

 Todet
ermi
nei
mpur
it
iespat
hway
sofdr
ugsubst
ancesanddr
ugpr
oduct
s(e.
g.,dur
ing

dev
elopmentphase)

 Toi
dent
if
yimpur
it
iesr
elat
edt
odr
ugsubst
ancesorexci
pient
s.

 Tounder
standt
hedr
ugmol
ecul
echemi
str
y

 To gener
ateai
mpur
it
iespr
ofi
l
ethatmi
micswhatwoul
dbe
obser
vedi
naf
ormalst
udyunderI
CHcondi
ti
ons.

1.
2.1LI
STOFRELATI
VEI
MPURI
TIES

Sl
no. Nameofi
mpur
it
ies

1 Fumar
icaci
d

2 n-
oxi
de

3 Di
l
uentPeak

4 I
mpur
it
y_J

5 I
mpur
it
y_Q

6 I
mpur
it
y_S

7 I
mpur
it
y_E

Appl
icat
ions
 Phar
maceut
icalAppl
icat
ions
1.Tocont
rol
impur
it
iesi
ndr
uganal
ysi
s.
2.Tabl
etdi
ssol
uti
onst
udyofphar
maceut
ical
dosagesf
orm.

R.
C.P.
H.S,
BERHAMPURPage14
 3.Phar
maceut
ical
qual
i
tycont
rol
.
 Env
ironment
alAppl
icat
ions
1.Det
ect
ionofphenol
i
ccompoundsi
ndr
inki
ngwat
er.
2.Bi
o-moni
tor
ingofpol
l
utant
s.
 Appl
icat
ionsi
nFor
ensi
cs
1.Quant
if
icat
ionofdr
ugsi
nbi
ologi
cal
sampl
es.
2.I
dent
if
icat
ionofst
eroi
dsi
nbl
ood,
uri
neet
c.

3.For
ensi
canal
ysi
soft
ext
il
edy
es.
4.Det
ermi
nat
ionofcocai
neandot
herdr
ugsofabusei
nbl
ood,
uri
ne
et
c.

 FoodandFl
avor
1.Measur
ementofQual
i
tyofsof
tdr
inksandwat
er.
2.Sugaranal
ysi
sinf
rui
tjui
ces.
3.Anal
ysi
sofpol
ycy
cli
ccompoundsi
nveget
abl
es.
4.Pr
eser
vat
iveanal
ysi
s.
 Appl
icat
ionsi
nCl
ini
calTest
s
1.Ur
ineanal
ysi
s,ant
ibi
oti
csanal
ysi
sinbl
ood.
2.Anal
ysi
sofbi
l
irubi
n,bi
l
iver
dini
nhepat
icdi
sor
der
s.
3.Det
ect
ionofendogenousNeur
opept
idesi
next
racel
l
ularf
lui
dofbr
ain
et
c.

Adv
ant
agesofUPLC:

 Wi
deappl
i
cabi
l
ity
 Hi
ghr
esol
uti
on
 Rapi
danal
ysi
s
 Hi
ghsensi
ti
vi
ty
 Excel
l
entr
epr
oduci
bil
i
ty
 Quant
it
ati
ve
 Easi
l
yaut
omat
ed

R.
C.P.
H.S,
BERHAMPURPage15
 1.
3 ANTI
PSYCOTI
CS

Ant
ipsy
chot
ics,al
soknownasneur
olept
icsormaj
ort
ranqui
li
zer
s,ar
e
a cl
ass ofmedi
cat
ion pr
imar
il
y used t
o manage psy
chosi
s(i
ncl
udi
ng
del
usi
ons,hal
l
uci
nat
ions,par
anoi
a ordi
sor
der
ed t
hought
),pr
inci
pal
l
yin
schi
zophr
eni
aandbi
pol
ardi
sor
der
.Ant
ipsy
chot
icsar
eusual
l
yef
fect
ivei
n
r
eli
evi
ngsy
mpt
omsofpsy
chosi
sint
heshor
tter
m.Thel
ong-
ter
m useof
ant
ipsy
chot
ics i
s associ
ated wi
th adv
erse ef
fect
s such as i
nvol
unt
ary
mov
ementdi
sor
der
s,gy
necomast
ia,i
mpot
ence,wei
ghtgai
nandmet
abol
i
c
sy
ndr
ome.

Fi
rst
-gener
ati
onant
ipsy
chot
ics,knownast
ypi
calant
ipsy
chot
ics,wer
e
di
scov
eredi
nthe1950s.Mostsecond-
gener
ati
ondr
ugs,knownasat
ypi
cal
ant
ipsy
chot
ics,
hav
ebeendev
elopedmor
erecent
ly,
alt
hought
hef
ir
stat
ypi
cal
ant
ipsy
chot
ic,cl
ozapi
ne,was di
scov
ered i
nthe 1960s and i
ntr
oduced
cl
i
nical
l
yint
he 1970s.
[
2]
Bot
h gener
ati
ons ofmedi
cat
ion t
end t
o bl
ock
r
ecept
orsi
nthebr
ain'
sdopami
nepat
hway
s

1.
3.1Mechani
sm ofact
ion

Ant
ipsy
chot
icdr
ugssuchashal
oper
idolandchl
orpr
omazi
net
endt
obl
ock
dopami
neD2 r
ecept
orsi
nthe dopami
ner
gicpat
hway
soft
he br
ain.Thi
s
meanst
hatdopami
ner
eleasedi
nthesepat
hway
shasl
essef
fect
.Excess
r
eleaseofdopami
nei
nthemesol
i
mbi
cpat
hwayhasbeenl
i
nkedt
opsy
chot
ic

R.
C.P.
H.S,
BERHAMPURPage16
exper
iences.Decr
easeddopami
ner
eleasei
nthepr
efr
ont
alcor
tex,
andexcess
dopami
ner
eleasei
not
herpat
hway
s,ar
eassoci
atedwi
thpsy
chot
icepi
sodes
i
n schi
zophr
eni
a and bi
pol
ardi
sor
der
.[
96]
[97]
I
n addi
ti
on t
otheant
agoni
sti
c
ef
fect
sofdopami
ne,ant
ipsy
chot
ics(
inpar
ti
cul
arat
ypi
calneur
olept
ics)al
so
ant
agoni
ze5-
HT2A r
ecept
ors.Di
ff
erental
l
elesoft
he5-
HT2A r
ecept
orhav
ebeen
associ
atedwi
thschi
zophr
eni
aandot
herpsy
choses,
incl
udi
ngdepr
essi
on.
[
98]
[99]

Hi
gherconcent
rat
ionsof5-
HT2A r
ecept
orsi
ncor
ti
calandsubcor
ti
calar
eas,
in
par
ti
cul
ari
nther
ightcaudat
enucl
eushav
ebeenhi
stor
ical
l
yrecor
ded.
[
98]

1.
3.2ADVERSEACTI
ON

 Sedat
ion(
par
ti
cul
arl
ycommonwi
thasenapi
ne,
clozapi
ne,
olanz
api
ne,
quet
iapi
ne,
chl
orpr
omaz
ineandz
ot ne[18])
epi
 Headaches
 Di
zzi
ness
 Di
arr
hea
 Anxi
ety
 Ext
rapy
rami
dal
sideef
fect
s(par
ti
cul
arl
ycommonwi
thf
ir
st-
gener
ati
on
ant
ipsy
chot
ics)
,whi
chi
ncl
ude:

-Akat
hisi
a—anof
tendi
str
essi
ngsenseofi
nnerr
est
lessness.

-Dy
stoni
a

-Par
kinsoni
sm

-Tr
emor

 Or
thost
ati
chy
pot
ensi
on
 Wei
ghtgai
n(par
ti
cul
arl
ypr
omi
nentwi
thcl
ozapi
ne,
olanzapi
ne,
quet
iapi
neand
zot ne[18])
epi

-Bl
urr
edv
isi
on
-Const
ipat
ion

R.
C.P.
H.S,
BERHAMPURPage17
 -Dr
ymout
h(al
thoughhy
per
sal
i
vat
ionmayal
sooccur
)

-Reducedper
spi
rat
ion.

R.
C.P.
H.S,
BERHAMPURPage18
DRUGPROFI
LE

2.
1QUETI NE10
API
BRANDNAME:
Ser
oquel
,Tempr
oli
de
STRUCTURE:

FI
G2.
1:Quet
iapi
ne
I
PUACNAME :
2-(
2-(
4-Di
benzo[
b,f
][1,
4]t
hai
zepi
ne-
11-
yl
-1-
pi
per
azi
nyl
)et
hoxy
)et
hanol
CATEGORY :
Ant
iPsy
cot
icdr
ug
SOLUBI
LITY : Fr
eel
ysol
ubl
e i
n Acet
oni
tr
il
e , wat
er and
met
hanol
butpar
ti
all
ysol
ubl
ein,
chl
orof
orm.
PHYSI
CALFORM :
Whi
teof
fpowder
MOLECULARFORMULA:
C21H25N3O2S

MOLECULARWEI
GHT :
383.
51g/
mol

CLASS :
Thai
zepi
ne
SUBCLASS :
Piper
izi
ne
HALFLI
FE :
21hr
s
MELTI
NGPOI
NT : 1760C
174-

DOSAGEFORMS,
COMPOSI
TIONANDPACKAGI
NG:
Quet
iapi
ne 150mg t
abl
etcont
ent
ing 150mg ofquet
iapi
ne and i
s
manuf
act
urebycompr
essi
ont
echni
que.Av
ail
abl
einauni
tdoseandbul
kby
st
ri
pandbot
tl
es.
ROUTEOFADMI
NISTRATI
ON:Or
al
VOLUMEOFDI
STRI
BITUTI
ON:
796±195L

2.
2PHARMACOKI
NETI
CPARAMETER
2.
2.1ABSORPTI
ON:

[
R.C.
P.H.
S,BERHAMPUR]
Page19
Or
alabsor
pti
onofquet
iapi
nei
svar
iabl
eandi
tisenhancebyf
oodandgast
ri
c
11
aci
d.bi
oav
ail
abi
l
ityi
s2-
5hr
saf
teradmi
nist
rat
ion.
2.
2.2DI
STRI
BUTI
ON:
Quet
iapi
nei
spr
otei
nboundi
nnat
ure.(
83%)
2.
2.3METABOLI
SM:
Quet
iapi
nei
smet
abol
i
sedbyLi
veri
ntomet
abol
i
tes.CYP3A4pl
ay
i
mpor
tantr
olei
nmet
abol
i
sm.Thepr
inci
palmet
abol
i
tei
snor
quet
iapi
newhi
ch
hasi
nvi
tr
oenemyofpar
asi
ti
cmov
ementt
hroughpl
asmacent
ral
i
zat
ionof
11
t
hesemet
abol
i
tesar
etwi
ceoft
hatquet
iapi
ne.
2.
2.4EXCREATI
ON:
Quet
iapi
neel
i
minat
ebyur
ine(
73%)andf
aces(
20%)
.r
enalexcr
eti
onof
11
nor
quet
iapi
nei
slesst
han3%ofi
.vdose.
2.
3PHARMACOLOGI
CALDATA
2.
3.1MOA:
Quet
iapi
nei
sadopami
ne,
ser
otoni
ne,
andadr
ener
gicant
agoni
st,
anda
pot
entant
ihi
stami
newi
thsomeant
ichol
i
ner
gicpr
oper
ti
es.Quet
iapi
nebi
nds
st
rongl
ytoser
otoni
nrecept
ors;t
hedr
ugact
saspar
ti
alagoni
stat5-
HT1A
r
ecept
ors.Ser
ialPET scans ev
aluat
ing t
he D2 r
ecept
oroccupancy of
quet
iapi
nehav
edemonst
rat
edt
hatquet
iapi
nev
eryr
api
dlydi
sassoci
atesf
rom
t
heD2 r
ecept
or.Theor
eti
cal
l
y,t
hisal
l
owsf
ornor
malphy
siol
ogi
calsur
gesof
dopami
net
o el
i
citnor
malef
fect
sin ar
eassuch ast
heni
grost
ri
ataland
t
uber
oinf
undi
bul
arpat
hway
s,t
husmi
nimi
zi
ngt
her
iskofsi
de-
eff
ect
ssuchas
pseudo-
par
kinsoni
sm as wel
las el
evat
ions i
n pr
olact
in.Some of t
he
ant
agoni
zedr
ecept
ors(
ser
otoni
n,nor
epi
nephr
ine)ar
eact
ual
l
yaut
orecept
ors
11
whosebl
ockadet
endst
oincr
easet
her
eleaseofneur
otr
ansmi
tt
ers.
12
2.
3.2USES:
Quet
iapi
neusei
ncur
ingofpsy
chol
ogi
cal
disease
Usedt
otr
eat
mentofsei
zur
e,mi
nordepr
essi
onandbi
pol
ardi
sease.
DEEFFECTS12
SI
Nausea
Vomi
ti
ng
Di
zzi
ness
CONTRAI
NDI ON12
CATI
Lowbl
oodpr
essur
e
[
R.C.
P.H.
S,BERHAMPUR]
Page20
 Tar
div
eDy
ski
nesi
a

[
R.C.
P.H.
S,BERHAMPUR]
Page21
LI
TERATUREREVI
EW
13
Pantetal
.,
(2012) Asi
mpl
e,accur
ateandpr
eci
seRP-
HPLCmet
hod
hasbeendev
elopedf
orest
imat
ionofQuet
iapi
neFumar
ate(
tabl
et)
.Thi
sassay
met
hodwasdev
elopedbyusi
ngcol
umnZor
baxODSC-
18,
150mm x4.
6mm,
5.
0µm asst
ati
onar
yphase.Buf
fer
:ACN(
65:
35)wasusedasmobi
l
ephase.
The% Assaywasf
oundi
nbet
ween98.
01-
98.
06.Thest
abi
l
itypar
amet
erof
t
hismet
hodwasdet
ermi
nedby
,cal
cul
ati
ngmeanper
cent
ager
ecov
ery
.The
Li
near
it
ywasf
oundt
obe0.
9999.

14
Kaushi
ketal
.,
(2018) hav
edev
elopedamet
hodf
orsi
mul
taneous
det
ermi
nat
ionofquet
iapi
neandi
tsmet
abol
i
tequet
iapi
nef
umar
atei
nhuman
pl
asma.Thi
smet
hoduseAt
lant
isdC18col
umn(
100mm×3.
0mm,3μm)at
40±5◦
C usi
ng a mobi
l
e phase mi
xtur
e(acet
oni
tr
il
e–met
hanol
-ammoni
um
acet
ate)
.Thesampl
ewasi
nject
edataf
lowr
ateof0.
4ml
/mi
n.andt
heel
uent
det
ect
edbyMS/
MSsy
stem byopt
imi
zi
ngm/
zrat
io.l
i
nearr
esponsebet
ween
concent
rat
ionandpeakar
ear
ati
ooft
hedr
ugi
nhumanpl
asmawasf
ound
ov
eraconcent
rat
ionr
angeof0.
2-600ng/
mland0.
4-600ng/
mlf
orQuet
iapi
ne
andQuet
iapi
nef
umar
ater
espect
ivel
y.Themeanr
ecov
eryofQuet
iapi
neand
Quet
iapi
nef
umar
atewer
efoundt
obe72.
96%&69.
3%r
espect
ivel
y.

15
Tal
usanietal
.,
(2013) Anewsi
mpl
e,sensi
ti
ve,
preci
seandaccur
ate
hi
ghper
for
mancel
i
qui
dchr
omat
ogr
aphymet
hodhasbeenest
abl
i
shedand
v
ali
dat
edf
oranal
ysi
sofQuet
iapi
nef
umar
atei
nbul
kandt
abl
etdosagef
orm.
Thesepar
ati
onwasachi
evedonaC18col
umnusi
ngami
xtur
eofphosphat
e
buf
fer
,acet
oni
tr
il
eandmet
hanoli
nther
ati
o50:
40:
10v
/v/
vwi
thaf
lowr
ateof
1ml
/mi
nanddet
ect
ionwav
elengt
hat245nm.
Ther
etent
iont
imeofQuet
iapi
ne
f
umar
atewasf
oundt
obeat5.
08mi
n.Li
near
it
yoft
hemet
hodwasf
oundt
o
bei
ntheconcent
rat
ionr
angeof10-
80µg/
mlwi
thcor
rel
ati
oncoef
fi
cientof
0.
999.Themet
hodwasv
ali
dat
edf
oraccur
acy
,pr
eci
sionandr
ecov
eryst
udi
es.
Li
mitofdet
ect
ion(
LOD)andl
i
mitofQuant
if
icat
ion(
LOQ)f
orQuet
iapi
newer
e
f
oundt
obe18.
815and57.
016µg/
mlr
espect
ivel
y.

16
Val
armat
hietal
.,
(2013) hav
edev
elopedaspect
rophot
omet
ri
c
anal
yti
calmet
hod f
ort
he qual
i
tycont
rolofQuet
iapi
ne Fumar
atei
ntwo

[
R.C.
P.H.
S,BERHAMPUR]
Page22
di
ff
erentcommer
cialmar
ket
edt
abl
etdosagef
orm(
BRANDA&BRANDB)of
samest
rengt
h25mg.Theabsor
bancedat
awasmeasur
edatawav
elengt
h
at290nm byusi
ngmet
hanol
:wat
eratar
ati
oof50:
50v
/v.Thel
i
near
it
ywas
f
oundatar
angeof16t
o24µg/
ml.Themeanr
ecov
eryoft
wobr
andswas
f
oundt
obe100.
41%t
o99.
77%.

17
Bagade et al
.,
(2009) Si
mpl
e, f
ast and r
eli
abl
e der
ivat
ive
Spect
rophot
omet
ri
cmet
hodswer
edev
elopedf
ordet
ermi
nat
ionofQuet
iapi
ne
f
umar
atei
nphar
maceut
icalf
ormul
ati
on.Secondor
derder
ivat
iveul
tr
avi
olet
Spect
rophot
omet
ri
c met
hods wer
e dev
eloped. Spect
rophot
omet
ri
cal
l
y,
Quet
iapi
ne f
umar
ate was det
ermi
ned by measur
ing t
he 2D-
val
ues at
254.
76nm wi
th0.
1N HClasbackgr
oundsol
vent
. Thel
i
near
it
y wi
thi
na
concent
rat
ionr
angef
rom 10t
o30μg/
ml.R.
S.D wasf
oundt
obe0.
20%
(
Quet
ipi
n®t
abl
et;
200mg)and0.
16%(
Quet
ipi
n®t
abl
et;
300mg)r
espect
ivel
y.
Theper
cent
ager
ecov
eri
eswer
enear100% f
orgi
venmet
hods.I
tcanbe
concl
udedt
hatt
hepr
oposedmet
hodsar
eful
l
yval
i
dat
edf
or2ndder
ivat
ive
scanandf
oundt
obesi
mpl
e,sensi
ti
ve,
accur
ate,
preci
se,
repr
oduci
ble,
rugged
andr
obustandr
elat
ivel
yinexpensi
ve.

18
Chandr
a etal
;
(2016) Ar
eli
abl
eand r
epr
oduci
bler
ever
sed-
phase
hi
ghper
for
mancel
i
qui
dchr
omat
ogr
aphy(
RP-
HPLC)wasdev
elopedf
ort
he
quant
it
ati
ve det
ermi
nat
ion ofQuet
iapi
ne Fumar
atef
rom mar
ket
ed bul
k
t
abl
ets.TheQuet
iapi
neFumar
atesepar
ati
onachi
evedwi
thC18col
umnusi
ng
t
hemet
hanolwat
ermobi
l
ephasei
nther
ati
o of30:
70 (
v/v
).Theact
ive
i
ngr
edi
entoft
hedr
ugcont
entquant
if
ywi
thUV det
ect
orat359nm.The
r
etent
iont
imeofQuet
iapi
neFumar
atei
s5.
27mi
n.Agoodl
i
near
it
yrel
ati
on
(
R2=0.
999)wasobt
ainedbet
weendr
ugconcent
rat
ionandav
eragepeakar
eas.
TheLOD andLOQ oft
hei
nst
rumentwer
ecal
cul
ated0.
02and0.
06µg/
mL,
r
espect
ivel
y.Theaccur
acyoft
hemet
hodv
ali
dat
ionwasdet
ermi
nedwi
tht
he
i
nter
-day(
100.
28%)andi
ntr
a-day(
100.
48%)r
ecov
eri
esoft
hedr
ug.The
quant
if
icat
ioncor
rel
ati
onr
angewas5-
50ppm.

19
Pr
asant
hetal
;(2011) Asi
mpl
e,sensi
ti
ve,
sel
ect
ive,
economi
caland
r
epr
oduci
ble UV spect
rophot
omet
ri
cmet
hod hasbeen dev
eloped f
ort
he
quant
it
ati
vedet
ermi
nat
ionofQTFi
nbul
kdr
ugandi
nphar
maceut
icaldosage

[
R.C.
P.H.
S,BERHAMPUR]
Page23
f
orms.The met
hodsar
e based on measur
ementofabsor
bance ofQTF
sol
uti
oni
net
hanolat207nm.Beer
’sl
awi
sobey
edov
ert
hel
i
nearr
ange1-
5
µg/
mL.Thepr
eci
sionr
esul
ts,expr
essedbyi
ntr
a-dayandi
nter
-dayr
elat
ive
st
andar
ddev
iat
ionv
alues,ar
esat
isf
act
oryi
.e% RSD100.
22% and99.
83%
r
espect
ivel
y.Theaccur
acyi
sal
sosat
isf
act
ory(
%RSD0.
39)andper
cent
age
r
ecov
eri
esar
eint
her
ange99.
34-100.
11%wi
tht
hest
andar
ddev
iat
ionof0.
39
.
TheLODandLOQwasf
ound0.
02and0.
12(
µg/
ml)
.

20
I
ngal
eetal
;(2013) Anew,
simpl
e,speci
fi
c,sensi
ti
ve,
rapi
d,accur
ate
andpr
eci
seRP-
HPLCmet
hodwasdev
elopedf
ort
heest
imat
ionofquet
iapi
ne
f
umar
atei
nbul
kandphar
maceut
icalf
ormul
ati
ons.Quet
iapi
nef
umar
atewas
chr
omat
ogr
aphedonMi
crosor
b-MV100-
5C-
18(
250x4.
6mm,5µm)col
umn
usi
ngUVdet
ect
or.Themobi
l
ephaseconsi
sti
ngacet
oni
tr
il
eandphosphat
e
buf
fer(
pH 3)i
nther
ati
oof50:
50(
v/v
)ataf
low r
ateof1.
0ml
/mi
nwi
th
det
ect
ion at292 nm wasused.Thel
i
near
it
ycur
ve wasobt
ained i
nthe
concent
rat
ionr
angeof5-
30µg/
ml.Theaccur
acyoft
hemet
hodwascar
ri
ed
out by addi
ng known amount of each dr
ug cor
respondi
ng t
othr
ee
concent
rat
ionl
evel
s80%,
100%and120%oft
hel
abel
clai
m.

21
Chhaj
ed etal
;(2009) New,si
mpl
e,costef
fect
ive,accur
ateand
r
epr
oduci
bleUV-
spect
rophot
omet
ri
cmet
hodsar
edev
elopedandv
ali
dat
edf
or
t
heest
imat
ionofquet
iapi
nef
umar
atei
nbul
kdr
ugandt
abl
etf
ormul
ati
on.
Quet
iapi
newasest
imat
edat239nm i
n0.
1Nhy
drochl
ori
caci
d(pH1.
2)andat
250nm i
net
hanol
.Beer
’sl
awwasobey
edi
ntheconcent
rat
ionr
angeof1–12
µgmL-
1(r
2=0.
9999)i
nhy
drochl
ori
caci
dand1–14µgmL-
1(r
2=0.
9998)i
n
t
heet
hanol
.Thedet
ect
ionandquant
it
ati
onl
i
mit
swer
efoundt
obe0.
0402,
0.
1217µgmL-
1inhy
drochl
ori
caci
dand0.
0384,0.
1163µgmL-
1inet
hanol
,
r
espect
ivel
y.

22
Nagar
aju etal
;
(2010) Two di
rect
,si
mpl
e,sensi
ti
ve and r
api
d
ext
ract
ion-
fr
eespect
rophot
omet
ri
cmet
hodshav
ebeen dev
eloped f
ort
he
det
ermi
nat
ionofquet
iapi
nef
umar
ate(
QTF)i
npur
efor
m andi
nit
sdosage
f
orms.Themet
hodsar
ebasedont
hef
ormat
ionofi
on-
pai
rcompl
exbet
ween
t
he dr
ug and t
wo sul
phont
hal
ein aci
dic dy
es,namel
y,br
omophenolbl
ue
(
met
hodA)andt
hymolbl
ue(
met
hodB)
,fol
l
owedbyt
hemeasur
ementof

[
R.C.
P.H.
S,BERHAMPUR]
Page24
absor
banceat410and380nm,
theper
cent
ager
ecov
eryv
alueswer
efoundt
o
be i
nthe r
ange of 97.
8–105.
6,wi
th st
andar
d dev
iat
ion v
alues f
rom
0.
88–1.
58%.

23
Rakshi
tetal
;
(2011) Thepr
esentwor
krepor
tsast
abi
l
ityi
ndi
cat
ing
r
ever
sedphaseul
tr
aper
for
mancel
i
qui
dchr
omat
ogr
aphy(
RP-
UPLC)met
hod
f
ort
hequant
it
ati
vedet
ermi
nat
ionofquet
iapi
nei
nphar
maceut
icaldosage
f
orm.Thechr
omat
ogr
aphi
csepar
ati
oni
sper
for
medonanAgi
l
entEcl
i
pse
Pl
usC18,
RRHD1.
8µm (
50mm x2.
1mm)col
umnusi
nggr
adi
entel
uti
on.The
opt
imi
zedmobi
l
ephaseconsi
stsof0.
1%aqueoust
ri
ethy
lami
ne(
pH7.
2)asa
sol
vent
-Aand80:
20v
/vmi
xtur
eofacet
oni
tr
il
eandmet
hanol
assol
vent
-B.The
el
utedcompoundsar
emoni
tor
edat252nm wav
elengt
husi
ngaUVdet
ect
or.
Ther
esponsewasf
oundl
i
nearf
orQUEf
rom 50% t
o150% ofst
andar
d
concent
rat
ionandcor
rel
ati
oncoef
fi
cientwasal
sof
oundgr
eat
ert
han0.
9999.

24
Pappul
a etal
;
(2015) Asi
mpl
e,r
api
d,accur
ateandpr
eci
seRP-
HPLC
met
hodwasdev
elopedf
ort
hedet
ermi
nat
ionofQuet
iapi
nef
umar
atei
npur
e
andt
abl
etdosagef
orms.Separ
ati
onoft
hedr
ugwasachi
evedonai
socr
ati
c
Shi
madzu pr
omi
nenceHPLC i
nst
rumenton aWat
ersXt
err
aC18 col
umn
(
250x4.
6mm,
5µ)
.Themet
hodshowedal
i
nearr
esponsef
orconcent
rat
ioni
n
t
her
angeof50–150μg/
mLusi
ngbuf
fer(
9.2±0.
05)andacet
oni
tr
il
eint
he
r
ati
oof51:
49v
/vwi
thdet
ect
ionat254nm wi
thaf
lowr
ateof1.
0mL/
minand
r
etent
iont
imewas6.
588mi
n.,t
he%RSDf
rom r
ecov
eryst
udi
eswasf
oundt
o
bel
esst
han1.

25
Saki
neetal
;
(2016) Anew hi
ghper
for
mancel
i
qui
dchr
omat
ogr
aphy
met
hodwasdev
elopedandv
ali
dat
edf
ort
hedet
ermi
nat
ionofquet
iapi
neand
i
tsmet
abol
i
tes7-
hydr
oxyquet
iapi
neandquet
iapi
nesul
foxi
dei
nratpl
asma.
Separ
ati
onwasper
for
medonaC18col
umn(
Zor
baxEcl
i
psePl
us4.
6mm
x100mm,
3.5µm par
ti
cles)usi
ngagr
adi
entel
uti
onataf
lowr
ateof1mL/
min.
Mobi
l
ephaseconsi
stedofacet
atebuf
fer(
10mM,pH5)andacet
oni
tr
il
e.
Anal
yteswer
edet
ect
edwi
thaDADdet
ect
orat225nm.Car
bamazepi
newas
usedasi
nter
nalst
andar
dinal
lanal
yses.I
twasf
oundt
obel
i
near
int
her
ange
of0.
065-
130µg/
mLf
orquet
iapi
ne,
0.086-
171µg/
mLf
or7-
hydr
oxyquet
iapi
ne

[
R.C.
P.H.
S,BERHAMPUR]
Page25
and0.
042-
83.
35µg/
mLf
orquet
iapi
nesul
foxi
de.

26
Bor
karetal
;(2009) Quet
iapi
nei
sthemostr
ecentagenti
ntr
oduced
ont
hedr
ugmar
ketf
ort
het
reat
mentofpsy
chot
icdi
sor
der
s.Onei
sthezer
o
or
derder
ivat
ivespect
roscopi
cmet
hod(
Met
hod-
I)andot
heri
sar
eaunder
cur
ve met
hod (
Met
hod-
II
),f
ort
he f
ir
stmet
hod,wav
elengt
h sel
ect
ed i
.e.
290.
0nm andt
hatoff
orot
her295.
0nm t
o281.
0nm r
espect
ivel
y.Pr
oposed
met
hodsgav
esat
isf
act
oryr
esul
tsi
nter
msofr
epeat
abi
l
ityandpr
eci
sioni
.e.%
RSD0.
60% and0.
73% r
esp.Al
soaccur
acyv
alueswer
ever
ygoodf
orbot
h
met
hodsi
.e.% RSD 0.
60and0.
75% r
esp.whi
chi
sdr
awnoutbyr
ecov
ery
st
udi
es,wer
efoundsat
isf
act
ory
.Bot
hspect
roscopi
cmet
hodshav
eexcel
l
ent
l
i
near
it
yandr
ange(
r2=/
0.999)
.Ruggednessofbot
hmet
hodschecksi
n
t
ermsofi
ntr
adayandi
nter
dayst
udi
eshav
ing%RSD0.
55%,
0.15%and0.
33%,
0.
46%r
espect
ivel
y.

27
Kor
rapol
u etal
;(2012) Thi
s st
udy descr
ibes dev
elopmentand
subsequentv
ali
dat
ionofast
abi
l
ityi
ndi
cat
ingr
ever
se-
phaseHPLCmet
hodf
or
t
he est
imat
ion ofQuet
iapi
ne Fumar
ate(
QF)i
n bul
k and phar
maceut
ical
dosagef
orms.Thepr
oposedRP-
HPLCmet
hodut
il
izesaI
ner
tsi
lODS(
250x
4.
6 mm,5 µ )col
umn,atambi
entt
emper
atur
e,opt
imum mobi
l
e phase
consi
sted of0.
02%v
/vf
ormi
c aci
d and met
hanol(
90:
10)
,ef
fl
uentf
low
moni
tor
edat1mL/
minandUVdet
ect
ionat220nm.Ther
etent
iont
imeofQF
was 13.
4mi
n.The l
i
near
it
ywas obser
ved i
nthe concent
rat
ion r
ange of
1050µg/
mLwi
thacor
rel
ati
oncoef
fi
cient(
R)of>0.
999.
Theper
centassayand
r
ecov
eryv
alueswer
efoundt
obei
nther
angeof98.
56-
99.
06% and99.
60-
100.
85%r
espect
ivel
y.

28
Pr
ati
k etal
;(2016) Toi
mpr
ovei
tsdi
ff
usi
onr
ate,i
twasembedded
i
ntol
i
posomaldi
sper
sion,whi
chhaspr
ovent
hati
thasadv
ancedef
fi
ciency
f
ordi
ff
usi
on.
Themet
hodusedf
ort
heest
imat
ionofQTFi
nbr
ainhomogenat
e
and pl
asma i
nvol
ved hi
gh-
per
for
mance l
i
qui
d chr
omat
ogr
aphy (
HPLC)

[
R.C.
P.H.
S,BERHAMPUR]
Page26
anal
ysi
s(ModelLC)usi
ngaC18col
umnwi
thUV det
ect
or.Mobi
l
ephase
consi
stedofphosphat
ebuf
fer(
pH3)
:acet
oni
tr
il
e:met
hanol(
50:
40:
10)andt
he
f
low r
atewas0.
8ml
/mi
n.Thesampl
evol
umei
nject
edwas20mlandt
he
wav
elengt
hwas247nm.
Liposomaldi
sper
siondet
ermi
nedas32.
61±1.
70l
ed
t
hesi
mpl
edi
sper
sionwi
thameasur
ementof27.
56±1.
33f
orper
centdr
ug
di
ff
usi
onat6h.

29
Sawsan et al
;(2015) Two chr
omat
ogr
aphi
c met
hods wer
e
dev
eloped f
ordet
ermi
nat
ionofquet
iapi
nef
umar
atei
npr
esenceoft
hree
r
elat
edcompounds;namel
yquet
iapi
neN-
oxi
de,des-
ethanolquet
iapi
neand
quet
iapi
nel
act
am,i
npur
efor
m andphar
maceut
icalpr
epar
ati
on.Thef
ir
st
met
hod depended ondensi
tomet
ri
cthi
nlay
erchr
omat
ogr
aphywher
ethe
separ
ati
onwasachi
evedusi
ngsi
l
icagel60F(
254)pl
atesasst
ati
onar
yphase
andt
oluene:
1,4-
dioxane:
dimet
hyl
ami
ne(
5:8:
2,v
/v/
v)asamobi
l
ephase.The
second met
hod ut
il
ized t
he r
ever
se phase hi
gh per
for
mance l
i
qui
d
chr
omat
ogr
aphi
ctechni
que,usi
ngC18col
umnandmet
hanol
:acet
oni
tr
il
e:
phosphat
ebuf
fer(
pH5.
3)i
nar
ati
o(19:
40:
41,v
/v/
v)asamobi
l
ephase.The
f
low r
atewas1mLmi
n-1andUV-
det
ect
ionwasatwav
elengt
h220nm.The
l
i
mitofLODandLOQwi
thi
nli
miti
.e,

30
Mahammadetal
;(2012) Theobj
ect
iveofpr
esentst
udywast
o
per
for
m someoft
hei
mpor
tantpr
efor
mul
ati
onst
udi
esofquet
iapi
nef
umar
ate
(
QPF)t
oobt
aint
henecessar
yinf
ormat
ionf
ort
hedesi
gnofcont
rol
l
eddr
ug
del
i
ver
ysy
stem.Thechr
omat
ogr
aphysepar
ati
onswer
ecar
ri
edoutonAgi
l
ent
ecl
i
pspl
usC-
8(4.
6X75mm,3.
5µm)col
umn.Thegr
adi
entmobi
l
ephase
consi
stsof10mM ammoni
um acet
ateandacet
oni
tr
il
e.Themobi
l
ephase
component
swer
efi
l
ter
edt
hrough0.
45µmembr
anef
il
terbef
oreuse.The
det
ect
ionwasmoni
tor
edat254nm andt
her
unt
imewas14mi
n.TheHPLC
anal
ysi
sresul
tsoft
hismi
xtur
eev
identofchemi
calst
abi
l
ityofQPFast
he
assaywaswi
thi
ntheaccept
abl
erange.

31
Rezaeietal
;(2018) Thegoaloft
hiswor
kwast
orecogni
zet
he
sy
nthesi
sandanal
yti
cal
def
erencemet
hodsofquet
iapi
nef
umar
ate.Apr
eci
se,
speci
fi
c, r
api
d and f
easi
ble r
ever
sed-
phase hi
gh-
per
for
mance l
i
qui
d

[
R.C.
P.H.
S,BERHAMPUR]
Page27
chr
omat
ogr
aphi
c(RP-
HPLC)
,UVspect
rophot
omet
ri
candr
ever
sedphaseul
tr
a
-
per
for
mance l
i
qui
d chr
omat
ogr
aphy (
RPUPLC) met
hods f
or t
he
det
ermi
nat
ion ofan ant
ipsy
chot
ic dr
ug quet
iapi
ne f
umar
ate has been
r
epor
ted.Theact
ivei
ngr
edi
entofquet
iapi
nef
umar
atesepar
ati
onachi
eved
wi
thC18col
umnusi
ngt
hemet
hanolwat
ermobi
l
ephasei
nther
ati
oof30:
70
(
v/v
).Theact
ivei
ngr
edi
entoft
hedr
ugcont
entquant
if
iesUVdet
ect
orat359
nm.Ther
etent
iont
imeofquet
iapi
nef
umar
atei
s5.
27mi
n.Agoodl
i
near
it
y
r
elat
ion(
r2=0.
999)wasobt
ainedbet
weendr
ugconcent
rat
ionandav
erage
peakar
eas.Thel
i
mitofdet
ect
ionandl
i
mitofquant
if
icat
ionoft
hei
nst
rument
wer
ecal
cul
ated0.
02and0.
06μg/
ml,
respect
ivel
y.Theaccur
acyoft
hemet
hod
v
ali
dat
ionwasdet
ermi
nedwi
tht
hei
nter
-day(
100.
28%)andi
ntr
a-day(
100.
48
%)r
ecov
eri
esoft
hedr
ug.Thequant
if
icat
ioncor
rel
ati
onr
angewas5-
50ppm.

32
Nahedetal;(
2009) st
udi
edv
olt
ammet
ri
cbehav
iorofQuet
iapi
ne
usi
ngdi
rectcur
rent
,di
ff
erent
ialpul
seandal
ter
nat
ingcur
rentpol
arogr
aphy
ov
ert
hepH r
angeof
6–11.
8andf
oundt
hatatpH 8,t
hedi
ff
usi
oncur
rent
concent
rat
ionr
elat
ionshi
pwasr
ect
il
inearov
ert
her
angeof8–44µg/
mLand
4–44µg/
mLusi
ngdi
rectcur
rentanddi
ff
erent
ialpul
sepol
arogr
aphymodes
r
espect
ivel
ywi
thami
nimum LODof0.
06µg/
mLand0.
04µg/
mLusi
ngdi
rect
cur
rentanddi
ff
erent
ial
pul
sepol
arogr
aphyr
espect
ivel
y.

[
R.C.
P.H.
S,BERHAMPUR]
Page28
RESEARCHENVI
SAGED

The I
nter
nat
ionalConf
erence on Har
moni
zat
ion (
ICH) gui
del
i
nes
ent
it
led‘
Stabi
l
ityTest
ingofNew Dr
ugSubst
ancesandPr
oduct
s’(
Q1A)R2
r
equi
ret
hatst
resst
est
ingshoul
dbecar
ri
edoutt
oel
uci
dat
ethei
nher
ent
st
abi
l
itychar
act
eri
sti
cs oft
he act
ive subst
ance.The gui
del
i
nes expl
i
cit
ly
r
equi
reconductoff
orceddecomposi
ti
onst
udi
esunderav
ari
etyofcondi
ti
ons,
l
i
kepH,
li
ght
,oxi
dat
ion,
dryheat
,et
c.andsepar
ati
onofdr
ugf
rom degr
adat
ion
pr
oduct
s.Themet
hodi
sexpect
edt
oal
l
owanal
ysi
sofi
ndi
vi
dualdegr
adat
ion
pr
oduct
s.Ar
evi
ew ofl
i
ter
atur
erev
eal
sal
argenumberofmet
hodsr
epor
ted
ov
ert
he per
iod ofl
ast3–4 decades undert
he nomencl
atur
e‘st
abi
l
ity-
i
ndi
cat
ing’
.Howev
er,mostoft
her
epor
tedmet
hodsf
allshor
tinmeet
ingt
he
cur
rentr
egul
ator
yrequi
rement
s.Accor
dingl
y,t
hepur
poseoft
hispr
ojectwor
k
i
stodev
elopav
ali
dat
edSt
abi
l
ityI
ndi
cat
ingAssayMet
hod(
SIAM)t
hatwi
l
l
meett
hecur
rentI
CHandr
egul
ator
yrequi
rement
sfort
hedr
ugsunderst
udy
.

Ext
ensi
vel
i
ter
atur
erev
iewr
eveal
edt
hatv
ari
ousmet
hodsl
i
keHPLC,
LC
-
MS/
MShav
ebeendev
elopedf
ort
heest
imat
ionofCar
vedi
l
olbutst
abi
l
ity
i
ndi
cat
ingmet
hodsar
enotav
ail
abl
efort
hesel
ect
eddr
ugs.

Hence,
anat
temptwasmadet
o:

 Dev
elopst
abi
l
ityi
ndi
cat
ingassaymet
hods

 I
tsOr
gani
cimpur
it
yst
udi
es.

[
R.C.
P.H.
S,BERHAMPUR]
Page29
5.
1:OBJECTI
VES
 Fr
om Li
ter
atur
erev
iew i
twasr
eveal
edt
hatst
rat
egi
eshav
e
beendev
elopedusi
ngHPLC,LCMS,andHPTLCbutnoUPLC
met
hod f
or Quet
iapi
ne and no Rel
ati
ve subst
ance f
or
Quet
iapi
nehasbeenr
epor
ted.
 Henceanat
tempti
smadet
o;
 Dev
elopUPLC assaymet
hodf
ort
heQuet
iapi
ne.
 Car
ryouti
tsr
elat
ivesubst
ancest
udy
.

5.
2:PLANEOFWORK

5.
2.1:Uv
-Vi
sSpect
rophot
omet
ri
cmet
hoddev
elopmentf
ort
heassessment
ofQuet
iapi
ne:
5.
2.1.
1:Opt
imi
zat
ion
 Tocar
ryoutsol
ubi
l
ityst
udi
esoft
hesel
ect
eddr
ug.
 Todet
ermi
newor
kingwav
elengt
hfort
hesel
ect
eddr
ugs.

5.
2.1.
2:Met
hod
 Topr
epar
est
ocksol
uti
onsoft
hesel
ect
eddr
ug.
 Topr
epar
ewor
kingst
andar
dsol
uti
onandconst
ructcal
i
brat
ion
cur
ves.

5.
2.2:UPLCmet
hoddev
elopmentf
ort
heest
imat
ionofQUETI
API
NE:
 Sel
ect
ionofMobi
l
ePhase.

 Pr
epar
ati
onofMobi
l
ePhase.

 Pr
epar
ati
onofSt
ockSol
uti
on.

 Cal
i
brat
ionCur
ves.

5.
3:RELATI
VESUBSTANCESTUDI
ES:
 Sel
ect
ionofMobi
l
ePhase.

[
R.C.
P.H.
S,BERHAMPUR]
Page30
  Pr
epar
ati
onofMobi
l
ePhase.

 Pr
epar
ati
onofSt
ockSol
uti
on.
 I
dent
if
icat
ionofr
elat
ivesubst
anc

[
R.C.
P.H.
S,BERHAMPUR]
Page31
EXPERI
MENTALWORK
7.
1ORGANOLEPTI
CCHARACTERI
STI
COFQUETI
API
NE
Col
our
:whi
teorof
fwhi
te
Odour :
char
act
eri
sti
c
Appear
ance :
powder
Tast
e :
tast
eless

7.
2.1UV-
VISspect
roscopi
cmet
hodf
ort
heest
imat
ionofQuet
iapi
ne
SOLUBI
LITY:
Sol
ubi
l
ityofdr
ugcar
ri
edoutusi
ngdi
ff
erentsol
ventl
i
keMet
hanol
,
Et
hanol
,Acet
oni
tr
il
e,Chl
orof
orm andWat
er.
Det
ermi
nat
ionofwor
kingwav
elengt
h:
Fordet
ermi
nat
ionofmaxi
mum wav
elengt
hofdr
ug,di
ff
erentconc.of
dr
ugsol
uti
onwer
epr
epar
edwi
thsol
venti
.e.met
hanolandwasscanni
ng
under200-
400nm wher
ecombi
nat
ionofMet
hanol
ist
akenasbl
ank.

7.
2.2METHOD
Pr
epar
ati
onofst
ocksol
uti
on:
St
ocksol
uti
onswer
epr
epar
edbydi
ssol
vi
ng10mgofdr
ugwi
th10ml
of
met
hanolt
oget1000ppm conc.Fr
om t
hatpr
epar
edsol
uti
ont
ake1mlof
sol
uti
onandadd10mlofsol
ventt
opr
epar
e100ppm conc.
,sol
uti
on.Fr
om
t
hat st
ock sol
uti
on we can t
ake 1,
2,
3,
4,
5ml r
espect
ivel
yto pr
epar
e
10,
20,
30,
40,
50µg/
mlconc.Sol
uti
on.

Cal
ibr
ati
onCur
ve:
Af
termaki
ngdi
ff
erentconc.oft
hepr
epar
edsol
uti
onwecangof
or
Beer
’sl
awpl
otf
orpur
edr
ugsoal
lpr
epar
edconc.Andscannedunder
200-
400
nm i
nUV-
spect
roscopyt
oobt
ainAbsor
banceandgr
aphwaspl
ott
edbyt
aki
ng
Absor
banceonY-
axi
sandConc.onX-
axi
s.

[
R.C.
P.H.
S,BERHAMPUR]
Page32
7.
3UPLCt
echni
quef
orest
imat
ionofQuet
iapi
ne
7.
3.1Met
hodOpt
imi
zat
ion
Chr
omat
ogr
aphycondi
ti
on
Fort
hisexper
iment
alwor
kthemobi
l
ephase(
A)wasacombi
nat
ionof
Acet
oni
tr
le:Amm.f
ormat
ebuf
ferpH 4.
6(8:
2)i
staken ataf
low 1ml
/mi
n.
Pur
gingi
sdonet
oremov
eifanypar
ti
cul
atemat
teri
spr
esentandf
il
ter
t
hrough0.
45µm membr
anef
il
ter
.Theni
nject
ionv
olumewas20µlandt
he
el
uentwasmoni
torat240nm.
7.
3.2Met
hoddev
elopment
Pr
epar
ati
onofBuf
fersol
uti
on
Buf
fersol
uti
onwaspr
epar
edbyt
aki
ng3.
87gm ofAmmoni
um acet
ateand
1gm of1-
oct
anesul
foni
caci
din1000mlbeakert
henpH wasadj
ust
edby
ammoni
asol
uti
ont
opH9.
5±0.
5.
Pr
epar
ati
onofmobi
lephase(
A)
Mobi
l
ephase(
A)waspr
epar
edbymi
xi
ngof950mlofBuf
fersol
uti
onand
50mlofMet
hanolt
hen soni
cat
ethemobi
l
ephasef
or10mi
ntor
emov
e
di
ssol
vedgases.
Pr
epar
ati
onofmobi
lephase(
B)
Mobil
ephase(B)waspr
eparedbymixi
ngof550mlofAcet
one,400mlofMethanol
and50mlofpuri
fi
edwat
erthensoni
cat
ethemobil
ephasefor10mint
or emove
di
ssol
vedgases.

Pr
epar
ati
onofst
andar
dsol
uti
on
10mgofQuet
iapi
nepowderdr
ugwasaccur
atel
ywei
ghtf
urt
her
mor
e,
t
akeni
na10mlv
olumet
ri
cfl
askt
oitt
hepr
epar
edmobi
l
ephasewasadded
whi
chr
esul
ted1000ppm.Fr
om t
hatst
ocksol
uti
onwecant
ake1mlsol
uti
on
andaddedt
hepr
epar
edmobi
l
ephasei
na10mlv
olumet
ri
cfl
askupt
omar
k
t
o get 100ppm.
From t
his di
ff
erent ppm. I
s pr
epar
e by t
aki
ng
0.
5,1,
1.5,
2.0,
2.5,
3.0,
3.5ml of sol
uti
on t
o get 5,
10,
15,
20,
25,
30,
35ppm
r
espect
ivel
y.
Cal
ibr
ati
onCur
ve
UPLCmet
hodf
orQuet
iapi
neTabl
ets
20t
abl
etsofQuet
iapi
newast
akenandwei
ghed,af
tert
hatt
het
abl
ets
wast
ri
tur
ated,f
rom t
hat57mgofpowderdr
ugwast
akenouti
nav
olumet
ri
c
f
lask,byt
hehel
pofabov
esol
ventt
hev
ol.wasmade-
up.Thent
hesol
n.i
s

[
R.C.
P.H.
S,BERHAMPUR]
Page33
t
akenf
orf
il
tr
ati
onand t
hef
il
tr
atewast
akent
o pr
epar
edesi
reconc.of
sol
uti
onandabov
esol
uti
ont
akenf
orUPLCanal
ysi
s.

7.
4UPLCf
orRel
ati
veSubst
ancesTest

Chr
omat
ogr
aphycondi
ti
on
Fort
hisexper
iment
alwor
kthemobi
l
ephase(
A)wasacombi
nat
ionof
Acet
oni
tr
le:Amm.
for
mat
e buf
fer pH 4.
6( 8:
2 )
ist
aken at a f
low
1ml
/mi
n.Pur
gingi
sdonet
oremov
eifanypar
ti
cul
atemat
teri
spr
esentand
f
il
tert
hrough0.
45µm membr
anef
il
ter.Theni
nject
ionv
olumewas20µland
t
heel
uentwasmoni
torat240nm.
Pr
epar
ati
onofBuf
fersol
uti
on
Buf
fersol
uti
onwaspr
epar
edbyt
aki
ng3.
87gm ofAmmoni
um acet
ateand
1gm of1-
oct
anesul
foni
caci
din1000mlbeakert
henpH wasadj
ust
edby
ammoni
asol
uti
ont
opH9.
5±0.
5.
Pr
epar
ati
onofmobi
lephase(
A)
Mobi
l
ephase(
A)waspr
epar
edbymi
xi
ngof950mlofBuf
fersol
uti
on
and50mlofMet
hanolt
hensoni
cat
ethemobi
l
ephasef
or10mi
ntor
emov
e
di
ssol
vedgases.
Pr
epar
ati
onofmobi
lephase(
B)
Mobi
l
ephase(
B)waspr
epar
edbymi
xi
ngof550ml
ofAcet
one,
400ml
of
Met
hanoland50mlofpur
if
iedwat
ert
hensoni
cat
ethemobi
l
ephasef
or
10mi
ntor
emov
edi
ssol
vedgases.
Pr
epar
ati
onofSt
andar
dSol
uti
on
About28mgofQuet
iapi
newasaccur
atel
ywei
ghedandt
ransf
ert
oa
v
olumet
ri
cfl
askandv
olumewasmakeupt
othemar
kwi
thdi
l
uent
s,soni
cat
e
f
or5mi
ns.Fur
therdi
l
uti
onwasmadebypi
pet
ti
ngout2mlsol
uti
ont
o100ml
v
olumet
ri
cfl
askandv
olumewasmakeupt
othemar
k.Fr
om t
hatsol
uti
on
agai
n2ml
wast
akent
omake20ml
sol
uti
on.
Pr
epar
ati
onofSy
stem Sui
tabi
li
tysol
uti
on
5mgofUSPQuet
iapi
nesy
stem sui
tabi
l
ityst
andar
dwasaccur
atel
ywei
ght
onpaper
,Tr
ansf
ert
o10mlv
olumet
ri
cfl
askandadd7mlofdi
l
uent
s,soni
cat
e
f
or15mi
nthent
hev
olumewasmakeupt
othemar
k.

[
R.C.
P.H.
S,BERHAMPUR]
Page34
[
R.C.
P.H.
S,BERHAMPUR]
Page35
RESULTANDDI
SCUSSI
ON
8.
1UV-
Visspect
rophot
omet
ri
cmet
hod
8.
1.1.Opt
imi
zat
ion
Sol
ubi
li
ty:Sol
ubi
l
ityofQuet
iapi
newascar
ryoutusi
ngv
ari
oussol
vent
sandt
he
r
esul
tswer
eshownbel
ow.
Tabl
e8.
1:Sol
ubi
li
tyofQuet
iapi
ne
SOLVENT DRUGSOLUBI
LITY

Di
st.
Wat
er I
nsol
ubl
e

Met
hanol Sol
ubl
e

Et
hanol Sol
ubl
e

Acet
oni
tr
il
e Sol
ubl
e

0.
1NHCL I
nsol
ubl
e

0.
1NNaOH I
nsol
ubl
e

Chl
orof
orm Par
ti
all
ySol
ubl
e

Hy
drogenPer
oxi
de I
nsol
ubl
e

8.
1.2Met
hod
Det
ermi
nat
ionofWor
kingWav
elengt
h
The dr
ug sol
uti
ons of Quet
iapi
ne wer
e scanned usi
ng UV-
VIS
spect
rophot
omet
erwi
thi
nthe wav
elengt
hregi
on of200-
400nm agai
nst
Met
hanolasbl
ank.Thewav
elengt
hofQuet
iapi
nei
s290nm.
Ther
esul
tsar
e
shownbel
owf
ig8.
1.

[
R.C.
P.H.
S,BERHAMPUR]
Page36
 Fi
g.8.
1:Ov
erl
ayUV-
spect
raofQuet
iapi
ne

Pr
epar
ati
onofcal
ibr
ati
oncur
ve:
Forpr
epar
ati
onofcal
i
brat
ioncur
veofQuet
iapi
ne,ast
d.sol
uti
onof
1000ppm waspr
epar
ed.ThenFr
om st
ocksol
uti
on,di
ff
erentconcent
rat
ion
wasmader
angi
ngf
rom 10-
50ppm wer
epr
epar
edandwasscannedusi
ngUV
-
VISspect
rophot
omet
er.Theconcent
rat
ionandcor
respondi
ngabsor
bance
wer
egi
veni
nthet
abl
ebel
ow.
Tabl
e8.
2:Cal
ibr
ati
ont
abl
eoft
heUV-
VISSpect
rophot
omet
ri
cmet
hodf
or
Quet
iapi
ne
CONC.(µg/
ml) ABSORBANCE

10 0.
181

20 0.
354

30 0.
501

40 0.
663

50 0.
829

Acal
i
brat
ioncur
vewaspl
otbyusi
ngt
heconcent
rat
iononX-
axi
sandmean
absor
banceonY-
axi
s.

[
R.C.
P.H.
S,BERHAMPUR]
Page37
Fi
g 8.
2:Cal
ibr
ati
on cur
ve ofQuet
iapi
ne by UV-
VIS spect
rophot
omet
ri
c
met
hod
Fr
om t
hecal
i
brat
ioncur
vei
twasf
oundt
hati
tshowsl
i
near
it
yint
her
angeof
10-
50µg/
mlwi
thr
egr
essi
oncoef
fi
cient0.
999.

8.
1.3Val
idat
ion
Tabl
e8.3:Accur
acyDat
aoft
heUV-
VisSpect
rophot
omet
ri
cMet
hodf
or
Quet
iapi
ne.

No of Amount of Amount of %Recov

ered %RSD
Pr
epar
ati
on Queti
apine Queti
api
ne
spi
ked(μg) Recover
ed(
μg)

80 80 79.
1 98.
2 0.
88

80 79.
1 98.
4

79 78.
9 99.
8

100 101 100.

2 99.
21 0.
66

100 100 100

99 99.
5 100.
51

120 121 120.

3 99.
54 0.
24

122 120.
6 99.
08

120 121.
9 99.
4

[
R.C.
P.H.
S,BERHAMPUR]
Page38
 Mean%r
ecov
ery 99.
35

Ov
eral
l%RSD 0.
74

Table8.4:Pr
eci
sionDat
aShowingRepeatabi
li
tyoft
heUV-
Vis.
Spectr
ophotometri
cMethodf
orQueti
apine.

Sl
.No Conc.
(g/
ml) Abs. Cal
c.Amt
. St
ati
sti
calAnal
ysi
s

1 20 0.
354 20.
61

2 20 0.
356 20.
74 Mean=20.
665

3 20 0.
355 20.
68 SD=0.
0641

4 20 0.
356 20.
74 %RSD=0.
310

5 20 0.
354 20.
61

6 20 0.
354 20.
61

Table8.
5:Intr
adayPr
eci
sionDat
aoft
heUV-
VisSpect
rophot
omet
ri
cMet
hod
forQuet
iapine

Sl
.No Conc.
(g/
ml) Abs.1 Abs.2 Abs.3 St
ati
sti
calAnal
ysi
s

1 20 0.
354 0.
356 0.
355

2 20 0.
352 0.
353 0.
354

3 20 0.
355 0.
354 0.
355
Mean=20.
65
4 20 0.
354 0.
356 0.
357
SD=0.
030
5 20 0.
356 0.
355 0.
354
%RSD=0.
147
6 20 0.
354 0.
355 0.
356

Mean 0.
3541 0.
3548 0.
3551

Cal
.Amt
.(g/
ml) 20.
62 20.
66 20.
68

[
R.C.
P.H.
S,BERHAMPUR]
Page39
Table8.
6:Inter
dayPr
eci
sionDat
aoft
heUV-
VisSpect
rophot
omet
ri
cMet
hod
forQuet
iapine

MeanAbsor
bance

Conc.
(g/
ml) Day1 Day2 Day3 St
ati
sti
calAnal
ysi
s

20 0.
354 0.
355 0.
354 Mean=20.
65

SD=0.
036

Cal
c.Amt
.(g/
ml) 20.
61 20.
68 20.
61 %RSD=0.
174

Tabl
e 8.
7:Ruggedness Dat
a oft
he oft
he UV-
Vis Spect
rophot
omet
ri
c
Met
hodbyDi
ff
erentAnal
yst
sforQuet
iapi
ne

Anal
yst
-1 Anal
yst
-2

Conc. Cal
c. Stat
isti
cal Conc. Cal
c. Stat
isti
cal
Amt. Analy
sis Amt. Analy
sis
(
g/
ml) Abs. (
g/
ml) Abs.

20 0.
354 20.
61 Mean 20 0.
355 20.
68 Mean=20.
65
=20.
676
20 0.
355 20.
68 20 0.
354 20.
61 SD=0.
053
SD=0.
058
20 0.
354 20.
61 20 20.
74 %RSD=0.
261
%RSD=0.
28 0.
356

20 0.
356 20.
74 20 0.
354 20.
61

20 0.
355 20.
68 20 0.
355 20.
68

20 0.
356 20.
74 20 0.
354 20.
61

Tabl
e 8.
8:Ruggedness Dat
a oft
he oft
he UV-
Vis Spect
rophot
omet
ri
c
Met
hodbyDi
ff
erentI
nst
rument
sforQuet
iapi
ne

UV-
1700 UV-
1800

[
R.C.
P.H.
S,BERHAMPUR]
Page40
Conc. Cal
c. Stat
isti
cal Conc. Cal
c. Stat
isti
cal
Amt. Analy
sis Amt. Analy
sis
(
g/
ml) Abs. (
g/
ml) Abs.

20 0.
355 20.
68 Mean 20 0.
354 20.
61
=20.
655
20 0.
354 20.
61 20 0.
355 20.
68 Mean=20.
67
SD=0.
053
20 0.
356 20.
74 20 0.
354 20.
61 SD=0.
058
%RSD
20 0.
354 20.
61 20 0.
356 20.
74 %RSD=0.
281
=0.
2611
20 0.
355 20.
68 20 0.
355 20.
68

20 0.
354 20.
61 20 0.
356 20.
74

Tabl
e8.9:RobustnessDat
aoftheoftheUV-VisSpect
rophot
omet
ri
cMet
hod
byDi
ffer
entSolventComposi
ti
onforQueti
apine

72:
28 78:
22

Conc. Cal
c. Stat
isti
cal Conc. Cal
c. Stat
isti
cal
Amt. Analy
sis Amt. Analy
sis
(
g/
ml) Abs. (
g/
ml) Abs.

20 0.
354 20.
61 Mean 20 0.
354 20.
61
=20.
67
20 0.
355 20.
68 20 0. 68 Mean=20.
355 20. 655
SD=0.
058
20 0.
354 20.
61 20 0.
354 20.
61 SD=0.
053
%RSD
20 0.
356 20.
74 =0.
281 20 0. 74 %RSD=0.
356 20. 2611

20 0.
355 20.
68 20 0.
355 20.
68

20 0.
356 20.
74 20 0.
354 20.
61

8.
2:ULTRAPERFORMANCELI
QUI
DCHROMATOGRAPHY(
UPLC)METHOD
8.
2.1:UPLCmet
hodf
orQuet
iapi
ne
8.
2.1.
1:Met
hodopt
imi
zat
ion
Tabl
e8.
10:Opt
imi
sedchr
omat
ogr
aphi
ccondi
ti
onofQuet
iapi
nef
orRP-
HPLC

[
R.C.
P.H.
S,BERHAMPUR]
Page41
met
hod
PARAMETER CONDI
TION

ACQUITYUPLC,
CSH
St
ati
onar
yphase(
col
umn)
C18,
2.1mm×100mm,17µm

Buf
fer:Met
hanol
(950:
50),
Mobi
l
ephase
ACN:Methanol
:
Water
(550:
400:
50)

Fl
owr
ate(
ml/
min) 0.
35ml
/mi
n

Runt
ime(
min) 15mi
n

Col
umnt
emper
atur
e(°C) 45

Vol
umeofi
nject
ion(
l) 2

Det
ect
ionwav
elengt
h(nm) 240nm

Dr
ugRt(
min) 4.
137

Tabl
e8.
11UPLCGr
adi
entMobi
lePhaseSol
uti
ons
Ti
me(
Min) %Mobi
lePhaseSol
uti
on %Mobi
lePhaseSol
uti
on
A B
0.
00 55 45
8.
00 55 45
8.
10 00 100
12.
00 00 100
12.
10 55 45
16.
00 55 45

[
R.C.
P.H.
S,BERHAMPUR]
Page42
8.
2.1.
2.Met
hodDev
elopment
1.Di
luent
s

Fi
g.8.
3:Repr
esent
ati
vechr
omat
ogr
am ofDi
luent
s
Her
enopeakwasf
ound.

2.St
andar
dsol
uti
on

Fi
g8.
4:Repr
esent
ati
vechr
omat
ogr
am ofSt
andar
dsol
uti
on
Her
eashar
ppeakwasobt
ainedat4.
137whi
chwast
akenast
hepeakof
st
andar
dsol
uti
on.

[
R.C.
P.H.
S,BERHAMPUR]
Page43
8.
3UPLCst
udyf
orRel
ati
veSubst
ances
8.
3.1Rel
ati
veSubst
ancesi
ndi
luent
s

Tabl
e8.
11:PeakResul
ts
Sl
.No Name RT Ar
ea %Ar
ea

1 Di
l
uentPeak_
1 0.
534 1815 1.
29

2 Di
l
uentPeak_
2 0.
713 1029 0.
73

3 Di
l
uentPeak_
3 0.
849 188 0.
13

4 Di
l
uentPeak_
4 1.
961 3195 2.
27

5 Di
l
uentPeak_
5 23.
533 9682 6.
88

6 Di
l
uentPeak_
6 23.
983 988 0.
71

7 Di
l
uentPeak_
7 24.
564 2409 1.
73

8 Di
l
uentPeak_
8 33.
328 1872 1.
35

9 Di
l
uentPeak_
9 34.
683 118291 85.
08

8.
3.2Rel
ati
veSubst
ancesi
nSy
stem Sui
tabi
li
tySol
uti
on

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Page44
Tabl
e8.
12:PeakResul
ts
Sl Name RT Ar
ea %Ar
ea Resol
uti
on
No.

1 Fumar
icAci
d 0.
573 667945 7.
26 NA

2 RC_
G 9.
157 100913 1.
10 39.
4

3 RC_
B 13.
636 112179 1.
22 15.
9

4 I
mpur
it
y- 14.
229 100719 1.
09 2.
1
1(
Deset
hoxy
)

5 Quet
iapi
ne 15.
946 8216721 89.
33 5.
8

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Page45
8.
3.3Rel
ati
veSubst
ancei
nPl
acebosol
uti
on

Tabl
e8.
13:PeakResul
ts
SlNo. Name RT Ar
ea %Ar
ea

1 Di
l
uentPeak_
1 0.
56 1105 0.
81

2 Di
l
uentPeak_
2 0.
716 866 0.
64

3 Di
l
uentPeak_
3 0.
853 422 0.
31

4 Di
l
uentPat
ter
n 1.
416 373 0.
28

5 Di
l
uentPeak_
4 1.
976 1010 0.
74

6 Quet
iapi
ne 15.
973 302 0.
22

7 Di
l
uentPeak_
5 23.
601 10100 7.
44

8 Di
l
uentPeak_
6 23.
983 942 0.
69

9 Di
l
uentPeak_
7 24.
568 2451 1.
81

10 Di
l
uentPeak_
8 33.
243 2027 1.
49

11 Di
l
uentPeak_
9 34.
717 116066 85.
55

8.
3.4Rel
ati
veSubst
ancei
nQuet
iapi
neTabl
ets25mg

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Page46
[
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Page47
Tabl
e8.
145:
PeakResul
ts

SlNo. Name RT Ar
ea %Ar
ea

1 Fumar
icAci
d 0.
568 1467176 7.
31

2 Di
l
uentPat
ter
n 1.
42 533 0.
00

3 Di
l
uentPeak_
4 1.
985 454 0.
00

4 Unk_
1 6.
207 4897 0.
02

5 N-
Oxi
de 6.
618 8903 0.
04

6 I
mp_
S 7.
243 2159 0.
01

7 Unk_
2 9.
963 6073 0.
03

8 I
mpur
it
y-Q 10.
897 1739 0.
01

9 RC_
B 13.
677 3762 0.
02

10 I
mpur
it
y-I
(Deset
hoxy
) 14.
220 4153 0.
02

11 Quet
iapi
ne 15.
915 18417943 91.
77

12 Unk_
3 16.
861 1932 0.
01

13 I
mpur
it
y_J 18.
359 7292 0.
04

14 Di
l
uentPeak_
5 23.
517 9895 0.
05

15 Di
l
uentPeak_
6 23.
983 1024 0.
01

16 Di
l
uentPeak_
7 24.
556 2525 0.
01

17 Unk_
4 25.
631 397 0.
00

18 Unk_
5 27.
365 381 0.
00

19 Unk_
6 28.
469 1763 0.
01

20 Unk_
7 28.
999 3884 0.
02

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Page48
21 I
mpur
it
y-E 29.
582 422 0.
00

22 Unk_
8 30.
101 422 0.
00

23 Unk_
9 30.
440 650 0.
00

24 I
mpur
it
y-W 30.
645 2310 0.
01

25 I
mpur
it
y-D 31.
276 1961 0.
01

26 Di
l
uentPeak_
8 33.
311 2137 0.
01

27 Di
l
uentPeak_
9 34.
717 114808 0.
57

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Page49
 CONCLUSI
ON
The obj
ect
ive ofI
CH gui
del
i
nes i
sto har
moni
ze i
nter
nat
ional
l
ythe
sci
ent
if
icnecessi
ti
est
omakesur
ethathar
mless,
successf
ul,
wit
hgoodcl
ass
medi
cinear
egener
atedatal
ow cost
.TheI
CH gui
del
i
newhi
chdeal
swi
th
“
steadi
nesschecki
ngofl
atestDr
ugandPr
oduct
s”pl
aysapi
vot
alr
olei
n
est
abl
i
shi
ng t
he st
abi
l
ity ofbul
k dr
ugs as wel
las t
he phar
maceut
ical
f
ormul
ati
ons.Her
etheai
mist
oemer
gentUPLCmet
hodf
orQuet
iapi
neandt
o
car
ryoutst
abi
l
ityst
udi
esatdi
ff
erentcondi
ti
onandi
tsr
elat
ivesubst
ances.
Fr
om t
he r
esul
ts ofmet
hod dev
elopmenti
twas f
ound t
hatt
he
dev
elopedmet
hodswer
esi
mpl
e,r
eli
abl
e,sensi
ti
veandaccur
ate.Quet
iapi
ne
show absor
pti
onmaxi
maat290nm i
nmet
hanoli
nUV spect
roscopy
.The
t
echni
que wast
o be l
i
nearwi
thi
nthe r
ange of10-
50ppm atr
egr
essi
on
coef
fi
cientof0.
999.
Theopt
imi
zedchr
omat
ogr
aphi
ccondi
ti
onsf
ort
hesel
ect
eddr
ugwer
ea
ACUI
TY CSH C-
18 col
umn,mobi
l
e phase(
A)was combi
nat
ion ofBuf
fer
:
Met
hanol(
950:
50)and mobi
l
ephase(
B)wascombi
nat
ion ofAcet
oni
tr
il
e:
Met
hanol
:Wat
er(
550:
450:
50)
,fl
ow speedbekeptupat0.
35ml
/mi
nand
el
uent
swer
echeckedat240nanomet
er.Thesampl
ewasi
nject
edbya2µl
i
nject
or.Al
lwer
eper
for
medatr
unt
imeof35mi
n.
Thet
echni
quewasobser
vedt
ober
api
d,easy
,consi
stent
,sensi
ti
ve,
cheapandcor
rect
.Thi
stechni
quewast
akenf
ort
hei
dent
if
icat
ionofdi
ff
erent
r
elat
ivesubst
anceswhi
chwer
epr
esenti
nthedi
l
uent
s,sy
stem sui
tabi
l
ity
sol
uti
on,pl
acebo sol
uti
on and st
andar
d sol
uti
on (
25mg)
.Some r
elat
ive
subst
anceswer
eident
if
ied.

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Page50
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