Pharmeuropa 29.01E

© Pharmeuropa 29.
1 | January 2017 1
Pharmeuropa archives
Texts for comment 29.1
2.2.32. Loss on drying .................................................................................................................. 2
2.3.2. Identification of fatty oils by thin-layer chromatography ...................................................... 4
5.23. Monographs on herbal drug extracts (information chapter) ................................................. 6
Akebia stem (2472)........................................................................................................................ 9
Ammonio methacrylate copolymer (type A) (2081)...................................................................... 14
Ammonio methacrylate copolymer (type B) (2082)...................................................................... 16
Arachis oil, hydrogenated (1171) ................................................................................................. 18
Arachis oil, refined (0263)............................................................................................................ 20
Bilberry fruit, dried (1588) ............................................................................................................ 22
Bilberry fruit, fresh (1602) ............................................................................................................ 25
Chlorobutanol (0382)................................................................................................................... 28
Chlorobutanol hemihydrate (0383) .............................................................................................. 32
Cinchocaine hydrochloride (1088) ............................................................................................... 36
Digitalis for homoeopathic preparations (2705) ........................................................................... 40
Ergocalciferol (0082) ................................................................................................................... 44
Erythropoietin concentrated solution (1316) ................................................................................ 50
Fenugreek (1323) ........................................................................................................................ 59
Flupentixol dihydrochloride (1693)............................................................................................... 61
Herbal drug extracts (0765) ......................................................................................................... 68
Isatis root (2566).......................................................................................................................... 74
Mepivacaine hydrochloride (1242)............................................................................................... 78
Milk thistle dry extract, refined and standardised (2071).............................................................. 84
Pentobarbital (0200) .................................................................................................................... 88
Pentobarbital sodium (0419)........................................................................................................ 93
Phenytoin sodium (0521)............................................................................................................. 98
Potassium sulfate (1622) ........................................................................................................... 103
Primaquine diphosphate (0635)................................................................................................. 105
Sodium sulfate, anhydrous (0099)............................................................................................. 110
Sodium sulfate decahydrate (0100)........................................................................................... 112
2 © Pharmeuropa 29.1 | January 2017
Reference: PA/PH/Exp. MG/T (16) 7 ANP
NOTE ON THE GENERAL CHAPTER

In view of its toxicity, the replacement of diphosphorus pentoxide with a molecular sieve is
proposed. The specialists in charge of the revision discussed the need to validate such a change
but came to the conclusion that no testing was required considering the inherent variability of
the current drying procedure and the experience gained with alternative drying agents. The
introduction of a standardised amount of desiccant to be used will also help to alleviate to a
certain extent that variability.
The conditions of ‘high vacuum’, which were reported to be difficult to achieve with available
instruments, are proposed for deletion ; this would trigger a revision of the concerned monographs
in due time.
Additional clarifications are proposed throughout the general chapter.
It also uses a new template for general chapters, which is under discussion within the on General
Methods (MG) Working Party, and any comments on this structure will be welcome.
XXXX:20232
2.2.32. LOSS ON DRYING

PRINCIPLE
Loss on drying is the loss of mass after drying under specified conditions, calculated expressed
as per cent m/m.
Drying to constant mass means that 2 consecutive weighings do not differ by more than 0.5 mg,
the 2nd weighing following an additional period of about 30 min of drying under the conditions
prescribed for the substance to be examined.
EQUIPMENT
The equipment typically consists of:
– weighing bottles that are made of suitable inert material and can easily be dried to constant
mass ; their diameter is large enough that the layer of the substance to be examined does
not exceed about 5 mm ;
– an analytical balance by which it is possible to determine reliably a change in mass of 0.1 mg ;
– depending on the procedure to be applied, a desiccator, a vacuum oven or an ordinary
laboratory oven ; in any case, the temperature of the oven is adjustable to the specified
temperature ± 2 °C ; vacuum ovens in which the pressure can be reduced to 1.5-2.5 kPa
are suitable ; ovens are qualified according to established quality system procedures, for
example by using a suitable certified reference material (amoxicillin trihydrate for performance
verification CRS may be used).
Equipment using other means of drying such as microwaves, halogen lamps, infrared lamps or
mixed technologies may be used provided they are demonstrated to be fit for purpose.
PROCEDURE
The substance to be examined must be homogenous and well mixed. If it contains large particles,
reduce the particle size by appropriate means.
It is recommended to perform the test in an environment that has minimal impact on sample
measurement (e.g. humidity).
Weigh Method. Place the prescribed quantity of the substance to be examined in a weighing
bottle that has been previously dried under the conditions prescribed for the substance to be
examined for at least 1 hour, and tared. Dry the substance to be examined to constant mass or for
the prescribed time by one of the following procedures, as required by the monograph. Where
the drying temperature is indicated by a single value rather than a range, drying is carried out at
the prescribed temperature ± 2 °C.
– a) “in In a desiccator” : the drying is carried out over diphosphorus pentoxide R about 100 g of
molecular sieve R at atmospheric pressure and at room temperature ;.
PA/PH/Exp. MG/T (16) 7 ANP

© Pharmeuropa 29.1 | January 2017 3
– b) “In vacuum (in vacuo) : the drying is carried out over diphosphorus pentoxide R about 100 g
of molecular sieve R at a pressure of 1.5 kPa to 2.5 kPa, at room temperature or within the
temperature range prescribed in the monograph ;.
– c) “in vacuo within a specified temperature range” : the drying is carried out over diphosphorus
pentoxide R, at a pressure of 1.5 kPa to 2.5 kPa within the temperature range prescribed in
the monograph ;
– d) “iIn an oven within a specified temperature range” : the drying is carried out in an oven within
the temperature range prescribed in the monograph ; instrument qualification is carried out
according to established quality system procedures, for example using a suitable certified
reference material (amoxicillin trihydrate for performance verification CRS may be used) ;.
– e) “under high vacuum” : the drying is carried out over diphosphorus pentoxide R at a pressure
not exceeding 0.1 kPa, at the temperature prescribed in the monograph.
If other conditions are prescribed, the procedure to be used is described in full in the monograph.
After drying in an oven, allow the weighing bottle and the sample to cool to room temperature in a
desiccator and weigh the weighing bottle containing the dried sample.
The mass of the sample is the difference between the mass of the filled weighing bottle and
the mass of the dried empty weighing bottle.
The loss on drying is the difference in the mass of the sample before and after drying, expressed
as a percentage, m/m being implicit.
Reagents
Molecular sieve. 1056600.

Molecular sieve composed of sodium aluminosilicate. It is available as beads with a pore size of
about 0.50.4 nm and with a diameter of about 2 mm.
PA/PH/Exp. MG/T (16) 7 ANP

Reference: PA/PH/Exp. 13H/T (16) 29 ANP

Method A (Figure 2.3.2.-1) : chromatogram of hydrogenated arachis oil added.
XXXX:20302
2.3.2. IDENTIFICATION OF FATTY OILS BY THIN-LAYER

CHROMATOGRAPHY
METHOD A
Thin-layer chromatography (2.2.27).
Test solution. Unless otherwise prescribed, dissolve about 20 mg (1 drop) of the fatty oil in 3 mL of
methylene chloride R.
Reference solution. Dissolve about 20 mg (1 drop) of maize oil R in 3 mL of methylene chloride R.
Plate : a suitable octadecylsilyl silica gel for high performance thin-layer chromatography as the
coating substance(1).
Mobile phase :
– mobile phase A : ether R ;
– mobile phase B : methylene chloride R, glacial acetic acid R, acetone R (20:40:50 V/V/V).
Application : 1 µL.
Development : twice over a path of 0.5 cm with mobile phase A, then twice over a path of 8 cm
with mobile phase B.
Drying : in air.
Detection : spray with a 100 g/L solution of phosphomolybdic acid R in ethanol (96 per cent) R.
Heat the plate at 120 °C for about 3 min and examine in daylight.
The chromatogram obtained typically shows spots comparable to those in Figure 2.3.2.-1.
1. arachis oil 5. soya-bean oil 9. sunflower oil 13. evening primrose oil
2. sesame oil 6. rapeseed oil (erucic 10. almond oil 14. safflower oil (type I)
acid-free)
3. maize oil 7. linseed oil 11. wheat-germ oil 15. safflower oil (type II)
4. rapeseed oil 8. olive oil 12. borage oil 16. hydrogenated arachis oil
Figure 2.3.2.-1. – Chromatograms for the identification of fatty oils (method A)
METHOD B
(1) Merck HPTLC Silica gel 60 RP-18 is suitable.
PA/PH/Exp. 13H/T (16) 29 ANP

Test solution. Unless otherwise prescribed, dissolve about 20 mg (1 drop) of the fatty oil in 3 mL of
methylene chloride R.
Reference solution. Dissolve about 20 mg (1 drop) of maize oil R in 3 mL of methylene chloride R.
Plate : a suitable octadecylsilyl silica gel for high performance thin-layer chromatography as the
coating substance(2).
Mobile phase : methylene chloride R, glacial acetic acid R, acetone R (20:40:50 V/V/V).
Application : 1 µL as bands of 8 mm. A suitable automated apparatus may be used.
Development : over a path of 7 cm.
Drying : in air.
Detection : treat with a 100 g/L solution of phosphomolybdic acid R in ethanol (96 per cent) R.
Heat the plate at 120 °C for 3 min and examine in daylight.
The chromatogram obtained typically shows zones comparable to those in Figure 2.3.2.-2.
1. arachis oil 5. soya-bean oil 9. sunflower oil 13. evening primrose oil
2. sesame oil 6. rapeseed oil (erucic 10. almond oil 14. safflower oil (type I)
acid-free)
3. maize oil 7. linseed oil 11. wheat-germ oil 15. safflower oil (type II)
4. rapeseed oil 8. olive oil 12. borage oil 16. hydrogenated arachis oil
Figure 2.3.2.-2. – Chromatograms for the identification of fatty oils (method B)
(2) Merck HPTLC Silica gel 60 RP-18 is suitable.

Reference: PA/PH/Exp. EXT/T (16) 3 ANP

Based on the distinction between DERgenuine and DERtotal, as proposed in the monograph Herbal
drug extracts (0765), published in the same issue of Pharmeuropa, the text has been amended
accordingly.
XXXX:52300
5.23. MONOGRAPHS ON HERBAL DRUG EXTRACTS

(INFORMATION CHAPTER)
Basis for elaboration of monographs on herbal drug extracts
European Pharmacopoeia monographs on herbal drug extracts are elaborated on the basis
of extracts present in medicinal products that have been authorised and/or registered by
the competent authorities of Parties to the Convention on the Elaboration of a European
Pharmacopoeia. However, these monographs do not necessarily cover all extracts which may
be available on the market.
European Pharmacopoeia monographs on herbal drug extracts are elaborated by groups
of experts and working parties in collaboration with national pharmacopoeia authorities, the
competent authorities for marketing authorisation, Official Medicines Control Laboratories
(OMCLs) and the EDQM laboratory ; they are also assisted by the producers of herbal drug
extracts and/or the pharmaceutical manufacturers that use these extracts.
During the elaboration of an extract monograph, the group of experts works on the basis of a
number of extracts from the specified herbal drug that are incorporated into authorised and/or
registered medicinal products originating from different sources. These extracts may have
been produced using different extraction solvents and/or extraction processes and may include
different types and contents of excipients (added for technological purposes during production
of the extract).
Where the results from the analysis of the extracts indicate that all of the extracts are in
compliance with all of the quality parameters, the monograph is intended to apply to all types of
production of that specific herbal extract (for example, Devil’s claw dry extract (1871), where
differences in the extraction solvent used, as stated in the Production section of the monograph,
and any differences between the extracts in terms of their production process have no significant
effect on the quality parameters).
Where there is a definable difference in the quality parameters between the extracts due to one
or more aspects of the production process, the monograph is presented as a family monograph
in which there will be a qualitative or quantitative difference applicable to one or more analytical
parameters (for example, Boldo leaf dry extract (1816), where differences in the extraction
solvents used, as stated in the Production section of the monograph, necessitates defining a
lower minimum content of assayed constituents in the aqueous extract in comparison with the
minimum content in the hydroalcoholic extract ; all other quality parameters are identical).
Where there is a significant difference in the quality parameters between the extracts due to one
or more aspects of the production process, more than one extract monograph is elaborated
(for example, Valerian dry aqueous extract (2400) and Valerian dry hydroalcoholic extract
(1898), where differences in the extraction solvents used and the processing method, as stated
in the Production section of the monographs, necessitates defining different minimum values
for the assayed constituents and different chromatographic profiles for the aqueous and the
hydroalcoholic extracts).
Types of extract
The general monograph Herbal drug extracts (0765) distinguishes different types of extracts.
This classification is based on the principles applied by European competent authorities during
the assessment of extracts in applications for marketing authorisation/registration of medicinal
products. These principles are summarised in Table 5.23.-1.
PA/PH/Exp. EXT/T (16) 3 ANP

This classification of extracts implies that, for each of these types of extract, distinct principles of
production and of defining or adjusting the content of assayed constituents are required by the
general monograph Herbal drug extracts (0765).
Genuine (native) extract. The concept of the genuine (native) drug extract ratio (DERgenuine) (see
the Glossary in the monograph Herbal drug extracts (0765)) was originally devised for application
to dry extracts where, after extraction of the herbal drug, all solvent is removed leaving only the
extracted dry matter from the herbal drug, that is, the genuine (native) extract. However, this
extracted dry matter often requires additives (inert excipients as processing aids) to produce a
technologically suitable extract. Such extracts made by different manufacturers from a given
herbal drug may have been produced using different solvents and processing methods and
contain different amounts of excipients relative to the quantity of extracted dry matter. In order to
permit comparison of such extracts, the declaration of the drug extract ratio (DER) on the basis of
the genuine (native) extract was introduced. Thus, for those dry extracts which can be produced
without excipients, the DER DERtotal and the DERgenuine are identical, but where excipients are
required, the DER DERtotal and the DERgenuine are different. It was then thought necessary to
apply this concept to soft extracts and liquid extraction preparations where solvent and, in some
cases, other substances are present as an integral part of the extract. For these extracts, the
concept of basing the DERgenuine on extracted dry matter was abandoned and the extract in its
entirety (including solvents, processing aids, etc.) is considered to be the genuine (native) extract.
Therefore, for these extracts, the DER DERtotal and the DERgenuine are identical. For the declaration
of the active substance in the finished product, only the DERgenuine is to be used.
Constituents for assay
For the purposes of quality control, monographs of the European Pharmacopoeia generally
include an assay. The choice of the constituent(s) to be assayed is linked, wherever possible, to
the regulatory process and is based upon the following criteria:
– where constituents with known therapeutic activity are present, these are selected for assay.
Constituents with known therapeutic activity are chemically defined substances or groups of
substances which are generally accepted to contribute substantially to the therapeutic activity
of a herbal drug, a herbal drug preparation or a herbal medicinal product.
– where constituents with known therapeutic activity are absent, marker constituents are
selected for assay.
Active markers are constituents or groups of constituents which are generally accepted to
contribute to the therapeutic activity of an extract.
Table 5.23.-1. – Classification and principles of production of extracts

Information available Extract concept
during assessment Quantitative parameters
as regards pharma- Extract
Quantity of genuine Extract adjustment
cological/therapeutic type
Constituent to be (native) extract that is
relevance of constitu-
analysed included in finished
ents
products
Constituents with known Standar- Constituents with Variable 1) By addition of inert
therapeutic activity dised known therapeutic excipients (dry extracts)
activity or solvents (liquid
Constant extraction preparations
or soft extracts)
2) By blending batches
Constituents which are Quantified Active marker Constant By blending batches
generally accepted Range
to contribute to the
therapeutic activity
Constituents chosen Other Analytical marker Constant None
solely for analytical Variable
purposes irrespective of
any pharmacological or
therapeutic activity which
they may be reported to
possess

Analytical markers are constituents or groups of constituents that serve solely for analytical
purposes, irrespective of any pharmacological or therapeutic activity which they may be
reported to possess.
Use of analytical markers in ‘other’ extracts
The assay method described in an individual monograph :
– is usually derived from the method used to assay the corresponding herbal drug ;
– is considered appropriate to determine the content of the chosen analytical marker ;
– serves as a general analytical platform for determining the content of the marker in the
individual extracts covered by the monograph for the purpose of quality control. The method
may also be suitable for stability studies ;
– may also be suitable as a basis for establishing assay methods to quantify the content of an
extract in a finished medicinal product.
The minimum content of the analytical marker when using the assay method :
– is established by assaying extracts used in authorised and registered medicinal products ;
– is given based on the final extract (genuine (native) extract with, where present, excipients) ;
– and thus :
– represents the minimum value found in the extracts used for establishing the monograph
after confirmation or modification following comments received from users, producers or
national authorities during the public enquiry ;
– is related to the minimum value as stated in the corresponding herbal drug monograph ;
– reflects the range of yields of that particular marker from the extraction process as well as
the stability of that marker during manufacturing ;
– is dependent on the amount of excipients added for technological reasons.
Since the monograph may encompass a broad range of extracts, the minimum value given for the
analytical marker is not regarded as a stand-alone quality criterion (as compared to other minimum
values given in the Pharmacopoeia). However, it gives an indication of the minimum values that
can be expected when producing an extract from a herbal drug compliant with its monograph.
Based on the information given above, manufacturers may be requested by competent authorities
to complement this information with a minimum value for their own extract depending on their
individual manufacturing process and excipients added.
NOTE : information on the characteristics of the extracts (for example, strength of extraction
solvent, percentage of genuine extract, etc.) analysed during the elaboration of an individual
extract monograph will be made available to users of the European Pharmacopoeia in the
Knowledge database on the EDQM website.

Reference: PA/PH/Exp. TCM/T (16) 143 ANP
NOTE ON THE MONOGRAPH
Identification B : illustration of powdered herbal drug introduced and its legend integrated into
text of Identification B.
XXXX:2472
AKEBIA STEM
Akebiae caulis
Mutong
木通
DEFINITION
Dried stem of Akebia quinata (Houtt.) Decne. or Akebia trifoliata (Thunb.) Koidz., or a mixture of
the 2 species.
Content : minimum 0.15 per cent of oleanolic acid (C30H48O3 ; Mr 456.7) (dried drug).
IDENTIFICATION
A. Cylindrical, often slightly twisted stem, 30-70 cm long and 0.5-2 cm in diameter. Externally
greyish-brown, rough, with irregular longitudinal cracks and furrows. Lenticels are distinct.
Nodes are sometimes swollen, with scars of lateral branches. The texture is light, compact and
easily broken. The fracture is uneven ; the bark is relatively broad, yellowish-brown, with pale
yellow, granular dots visible ; the wood is yellowish-white, with radial striations, the pith is small,
occasionally hollowed, yellowish-white or yellowish-brown.
B. Microscopic examination (2.8.23). The powder is pale yellow. Examine under a microscope
using chloral hydrate solution R. The powder shows the following diagnostic characters :
numerous groups of fibres with crystal sheaths, 1-2.5 mm long, often in bundles ; parenchyma
cells containing isolated cubic or prismatic crystals of calcium oxalate ; golden-yellow,
tetragonal, polygonal or rounded sclereids, 25-50 µm in diameter, with very thick walls
and conspicuous pits ; vessels, mostly bordered-pitted, up to 120-160 µm ; crystals usually
polyhedral, often prisms ; reddish-brown polygonal cork cells with brown contents ; lignified
parenchyma cells with very thick, pitted walls.
B. Microscopic examination (2.8.23). The powder is pale yellow. Examine under a microscope
using chloral hydrate solution R. The powder shows the following diagnostic characters (Figure
2472.-1) : numerous groups of fibres, frequently broken [C], whose narrow lumens contain
small prisms of calcium oxalate [Ca] ; these groups are usually surrounded by crystal sheaths
of calcium oxalate [Cb] ; groups of parenchymatous cells with thick, pitted walls [F], some
containing compact masses of large polyhedral or prismatic crystals of calcium oxalate [Fa] ;
some of these cells are isolated [G]; cork fragments consisting of reddish-brown polygonal
cells with brown contents (surface view [A]) ; fragments of parenchyma consisting of cells with
thin colourless walls (transverse section [K], longitudinal section [L]) ; isolated cubic or prismatic
crystals of calcium oxalate [D] ; tetragonal, polygonal or rounded sclereids with very thick walls
and granular contents [B, M] ; narrow tracheids with pits and very fine oblique lines [E] and
vessels, mostly bordered pitted, up to 120-160 µm [J] ; ligneous parenchyma cells with very
thick, pitted walls and more or less rectangular medullary rays [H].
PA/PH/Exp. TCM/T (16) 143 ANP

Figure 2472.-1. – Illustration for identification test B of powdered herbal drug of akebia stem
C. Thin-layer chromatography (2.2.27).
Test solution. To 0.5 g of the coarsely powdered herbal drug (1400) (2.9.12) add 5 mL of
methanol R. Sonicate for 10 min and filter or centrifuge. Use the filtrate or the supernatant
as the test solution.
Reference solution. Dissolve 1 mg of hederagenin R and 1 mg of oleanolic acid R in 8 mL

of methanol R.
Plate : TLC silica gel plate R (2-10 µm)(3).
Mobile phase : glacial acetic acid R, acetone R, toluene R (5:20:80 V/V/V).
Application : 15 µL as bands of 8 mm.
Drying : in air.
Detection : treat with a 10 per cent V/V solution of sulfuric acid R in ethanol 96 per cent R, heat
at 105 °C until the spots appear and examine in daylight.
Results : see below the sequence of zones present in the chromatograms obtained with the
reference solution and the test solution. Furthermore, other coloured zones may be present in
the chromatogram obtained with the test solution.
(3) Merck HPTLC Si 60 F254 20 ×10 cm is suitable.

Top of the plate

A faint purple-blue zone
_______ _______
A purple-blue zone
Oleanolic acid : a pink zone A faint pink zone (oleanolic acid)
Sequence of narrow purple-blue zones
Hederagenin : a pinkish-blue zone
A prominent purple-blue zone
_______ _______
Reference solution Test solution
TESTS
Total ash (2.4.16) : maximum 6.5 per cent.
Loss on drying (2.2.32) : maximum 10.0 per cent, determined on 1.000 g of the powdered herbal
drug (355) (2.9.12) by drying in an oven at 105 °C for 3 h.
Aristolochic acids (2.8.21, Method A). It complies with the test.
ASSAY
The following chromatogram is shown for information but will not be published in the European
Pharmacopoeia.
1. oleanolic acid
Figure 2472.-2. – Chromatogram for the assay of akebia stem : test solution

Pharmacopoeia.
1. oleanolic acid
Figure 2472.-3. – Chromatogram for the assay of akebia stem : reference solution (a)
Pharmacopoeia.
1. oleanolic acid 2. ursolic acid
Figure 2472.-4. – Chromatogram for the assay of akebia stem : reference solution (b)
Liquid chromatography (2.2.29).
Test solution. To 2.00 g of the coarsely powdered herbal drug (1400) (2.9.12) add 50 mL of
methanol R, sonicate for 30 min and filter. Wash the residue with an appropriate quantity of
methanol R, combine the filtrate and washings and evaporate to dryness. Dissolve the residue in
10 mL of water R, extract with 3 quantities, each of 20 mL, of butanol R saturated with water R,
combine the extracts and evaporate to dryness. Take up the residue with a mixture of 2 mL of
hydrochloric acid R and 20 mL of methanol R and heat under a reflux condenser for 2 h. Add
10 mL of water R, extract with 2 quantities, each of 20 mL, of methylene chloride R, combine the
extracts and evaporate to dryness. Dissolve the residue in methanol R and dilute to 10.0 mL
with the same solvent.
Reference solution (a). Dissolve 20.0 mg of oleanolic acid CRS in methanol R and dilute to
20.0 mL with the same solvent.
Reference solution (b). Dissolve 10 mg of ursolic acid R in reference solution (a) and dilute to
10.0 mL with the same solution.
Reference solutions (c), (d), (e), (f), (g), (h). Dilute reference solution (a) with methanol R to
obtain 6 reference solutions of oleanolic acid, the concentrations of which span the expected
value in the test solution.

Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for chromatography R (5 µm)(4).
Mobile phase : triethylamine R, glacial acetic acid R, water R, methanol R (0.02:0.04:13:87 V/V/V/V).
Flow rate : 1.0 mL/min.
Detection : evaporative light-scattering detector ; the following settings have been found to be
suitable ; if the detector has different setting parameters, adjust the detector settings so as to
comply with the system suitability criterion for signal-to-noise ratio :
– carrier gas : nitrogen R ;
– pressure : 350 kPa ;
– evaporator temperature : 60 °C.
Injection : 10 µL.
Run time : 24 min.
Retention time : oleanic acid = about 19 min ; ursolic acid = about 20 min.
System suitability :
– resolution : minimum 1.7 between the peaks due to oleanolic acid and ursolic acid in the
chromatogram obtained with reference solution (b) ;
– signal-to-noise ratio : minimum 40 for the peak due to oleanolic acid in the chromatogram
obtained with reference solution (a).
Establish a calibration curve with the logarithm of the concentration (in milligrams per 10 mL) of
reference solutions (c), (d), (e), (f), (g) and (h) (corrected by the declared percentage content of
oleanolic acid CRS) as the abscissa and the logarithm of the corresponding peak area as the
ordinate.
Calculate the percentage content of oleanolic acid using the following expression :
A = logarithm of the concentration of oleanolic acid in the test solution, determined

from the calibration curve ;
m = mass of the herbal drug to be examined used to prepare the test solution, in
grams.
(4) Waters Symmetry C18 is suitable.


Identification : reference spectrum replaced by reference substance, in accordance with current
policy.
Assay : anhydrous formic acid R and acetic anhydride R have been replaced by a solution of
copper acetate R in glacial acetic acid R ; the proposed method has been shown to be more
robust than the current method.
XXXX:2081
AMMONIO METHACRYLATE COPOLYMER (TYPE A)
Ammonio methacrylatis copolymerum A
DEFINITION
Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-2-(trimethylammonio)ethyl
2-methylpropenoate) chloride having a mean relative molecular mass of about 150 000.
The ratio of ethyl propenoate groups to methyl 2-methylpropenoate groups to
2-(trimethylammonio)ethyl 2-methylpropenoate groups is about 1:2:0.2.
Content of ammonio methacrylate groups : 8.9 per cent to 12.3 per cent (dried substance).
CHARACTERS
Appearance : colourless to white or almost white granules or powder.
Solubility : practically insoluble in water, freely soluble in anhydrous ethanol and in methylene
chloride giving clear to cloudy solutions. Due to the polymeric nature of the substance, a stirring
time of up to 5 h may be necessary.
IDENTIFICATION
A. Infrared absorption spectrophotometry (2.2.24).
Comparison : Ph. Eur. reference spectrum of ammonio methacrylate copolymer (type A)
ammonio methacrylate copolymer CRS.
B. Viscosity (see Tests).
C. It complies with the limits of the assay.
TESTS
Solution S. Dissolve a quantity of the substance to be examined corresponding to 12.5 g of the
dried substance in a mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R.
Viscosity (2.2.10) : maximum 15 mPa·s, determined on solution S.
Apparatus : rotating viscometer.
Dimensions :
– spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft diameter = 4.0 mm ;
– cylinder : diameter = 27.62 mm ; height = 0.135 m.
Stirring speed : 30 r/min.
Volume of solution : 16 mL of solution S.
Temperature : 20 °C.

Appearance of a film. Spread 2 mL of solution S evenly on a glass plate. Upon drying a clear
film is formed.
Monomers. Liquid chromatography (2.2.29).
Solution A. Dissolve 3.5 g of sodium perchlorate R in water for chromatography R and dilute to
100 mL with the same solvent.
Test solution. Dissolve 5.00 g of the substance to be examined in methanol R and dilute to
50.0 mL with the same solvent. To 10.0 mL of this solution add 5.0 mL of solution A, dropwise,
while continuously stirring. Remove the precipitated polymer by centrifugation. Use the clear
supernatant solution.
Reference solution. Dissolve 50.0 mg of ethyl acrylate R and 10.0 mg of methyl methacrylate R in
methanol R and dilute to 50.0 mL with the same solvent. Dilute 1.0 mL of the solution to 100.0 mL
with methanol R. Add 10 mL of this solution to 5 mL of solution A.
Column :
– size : l = 0.12 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for chromatography R (7 µm)(5).
Mobile phase : dilute phosphoric acid R with water for chromatography R to obtain a solution at
pH 2.0 ; mix 800 mL of this solution and 200 mL of methanol R, filter and degas.
Detection : spectrophotometer at 202 nm.
Injection : 50 µL.
System suitability : reference solution :
– resolution : minimum 1.5 between the peaks due to impurity A and impurity B.
Limits :
– impurity A : not more than the area of the corresponding peak in the chromatogram obtained
with the reference solution (100 ppm) ;
– impurity B : not more than 2.5 times the area of the corresponding peak in the chromatogram
obtained with the reference solution (50 ppm).
Methanol (2.4.24, System A) : maximum 1.5 per cent.
Loss on drying (2.2.32) : maximum 3.0 per cent, determined on 1.000 g by drying in vacuo
at 80 °C for 5 h.
ASSAY
Dissolve 1.000 g in a mixture of 3 mL of anhydrous formic acid R and 30 mL of anhydrous acetic
acid R and heat to dissolve. Add 20 mL of acetic anhydride R. 75 mL of glacial acetic acid R at
about 50 °C within about 30 min. Allow to cool to room temperature and add 25 mL of a 6 g/L
solution of copper acetate R in glacial acetic acid R. Titrate with 0.1 M perchloric acid, determining
the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg of C9H18O2NCl (ammonio methacrylate
groups).
IMPURITIES
Specified impurities : A, B.
A. ethyl propenoate (ethyl acrylate),
B. methyl 2-methylpropenoate (methyl methacrylate).
(5) Polygosil 60-7, C18 is suitable.


Identification : reference spectrum replaced by reference substance, in accordance with current
policy.
Assay : anhydrous formic acid R and acetic anhydride R have been replaced by a solution of
copper acetate R in glacial acetic acid R ; the proposed method has been shown to be more
robust than the current method.
XXXX:2082
AMMONIO METHACRYLATE COPOLYMER (TYPE B)
Ammonio methacrylatis copolymerum B
DEFINITION
Poly(ethyl propenoate-co-methyl 2-methylpropenoate-co-2-(trimethylammonio)ethyl
2-methylpropenoate) chloride having a mean relative molecular mass of about 150 000.
The ratio of ethyl propenoate groups to methyl 2-methylpropenoate groups to
2-(trimethylammonio)ethyl 2-methylpropenoate groups is about 1:2:0.1.
Content of ammonio methacrylate groups : 4.5 per cent to 7.0 per cent (dried substance).
CHARACTERS
Appearance : colourless to white or almost white granules or powder.
Solubility : practically insoluble in water, freely soluble in anhydrous ethanol and in methylene
chloride giving clear to cloudy solutions. Due to the polymeric nature of the substance, a stirring
time of up to 5 h may be necessary.
IDENTIFICATION
Comparison : Ph. Eur. reference spectrum of ammonio methacrylate copolymer (type B)
ammonio methacrylate copolymer CRS.
B. Viscosity (see Tests).
C. It complies with the limits of the assay.
TESTS
Solution S. Dissolve a quantity of the substance to be examined corresponding to 12.5 g of the
dried substance in a mixture of 35.0 g of acetone R and 52.5 g of 2-propanol R.
Viscosity (2.2.10) : maximum 15 mPa·s, determined on solution S.
Apparatus : rotating viscometer.
Dimensions :
– spindle : diameter = 25.15 mm ; height = 90.74 mm ; shaft diameter = 4.0 mm ;
– cylinder : diameter = 27.62 mm ; height = 0.135 m.
Stirring speed : 30 r/min.
Volume of solution : 16 mL of solution S.

Appearance of a film. Spread 2 mL of solution S evenly on a glass plate. Upon drying a clear
film is formed.
Monomers. Liquid chromatography (2.2.29).
Solution A. Dissolve 3.5 g of sodium perchlorate R in water for chromatography R and dilute to
Test solution. Dissolve 5.00 g of the substance to be examined in methanol R and dilute to
50.0 mL with the same solvent. To 10.0 mL of this solution add 5.0 mL of solution A, dropwise,
while continuously stirring. Remove the precipitated polymer by centrifugation. Use the clear
supernatant solution.
Reference solution. Dissolve 50.0 mg of ethyl acrylate R and 10.0 mg of methyl methacrylate R in
methanol R and dilute to 50.0 mL with the same solvent. Dilute 1.0 mL of the solution to 100.0 mL
with methanol R. Add 10 mL of this solution to 5 mL of solution A.
Column :
– size : l = 0.12 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for chromatography R (7 µm).
Mobile phase : dilute phosphoric acid R with water for chromatography R to obtain a solution at
pH 2.0 ; mix 800 mL of this solution and 200 mL of methanol R, filter and degas.
Injection : 50 µL.
– resolution : minimum 1.5 between the peaks due to impurity A and impurity B.
Limits :
– impurity A : not more than the area of the corresponding peak in the chromatogram obtained
with the reference solution (100 ppm) ;
– impurity B : not more than 2.5 times the area of the corresponding peak in the chromatogram
obtained with the reference solution (50 ppm).
Methanol (2.4.24, System A) : maximum 1.5 per cent.
at 80 °C for 5 h.
ASSAY
Dissolve 2.000 g in a mixture of 3 mL of anhydrous formic acid R and 30 mL of anhydrous acetic
acid R and heat to dissolve. Add 20 mL of acetic anhydride R. 75 mL of glacial acetic acid R at
about 50 °C within about 30 min. Allow to cool to room temperature and add 25 mL of a 6 g/L
solution of copper acetate R in glacial acetic acid R. Titrate with 0.1 M perchloric acid, determining
the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 20.77 mg of C9H18O2NCl (ammonio methacrylate
groups).
IMPURITIES
Specified impurities : A, B.
A. ethyl propenoate (ethyl acrylate),
B. methyl 2-methylpropenoate (methyl methacrylate).


Identification : reference to the test for composition of fatty acids has been moved to the first
identification since it is more specific than the identification of fatty oils by TLC ; the latter has been
moved to the second identification.
XXXX:1171
ARACHIS OIL, HYDROGENATED
Arachidis oleum hydrogenatum

DEFINITION
Oil obtained by refining, bleaching, hydrogenating and deodorising oil obtained from the shelled
seeds of Arachis hypogaea L. Each type of hydrogenated arachis oil is characterised by its
nominal drop point.
CHARACTERS
Appearance : white or faintly yellowish, soft mass which melts to a clear, pale yellow liquid when
heated.
Solubility : practically insoluble in water, freely soluble in methylene chloride and in light petroleum
(bp : 65-70 °C), very slightly soluble in ethanol (96 per cent).
IDENTIFICATION
First identification : A, B, C.
Second identification : A, C, B.
A. Drop point (see Tests).
B. Identification of fatty oils by thin-layer chromatography (2.3.2).
Results : the chromatogram obtained is similar to the chromatogram for arachis oil shown in
Figure 2.3.2.-1.
C. Composition of fatty acids (see Tests).
TESTS
Drop point (2.2.17) : 32 °C to 43 °C, and within 3 °C of the nominal value.
Acid value (2.5.1) : maximum 0.5.
Dissolve 10.0 g in 50 mL of the prescribed solvent by heating on a water-bath.
Peroxide value (2.5.5, Method A) : maximum 5.0.
Dissolve 5.0 g in 30 mL of the prescribed solvent by heating on a water-bath.
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent.
Alkaline impurities (2.4.19). It complies with the test.
Composition of fatty acids (2.4.22, Method A). Use the mixture of calibrating substances in
Table 2.4.22.-3.
Column :
– material : fused silica ;
– size : l = 25 m, Ø = 0.25 mm ;
– stationary phase : poly(cyanopropyl)siloxane R (film thickness 0.2 µm).
Carrier gas : helium for chromatography R.
Split ratio : 1:100.
Temperature :
– column : 180 °C for 20 min ;
– injection port and detector : 250 °C.

Detection : flame ionisation.

Composition of the fatty-acid fraction of the oil :
– saturated fatty acids of chain length less than C14 : maximum 0.5 per cent ;
– myristic acid : maximum 0.5 per cent ;
– palmitic acid : 7.0 per cent to 16.0 per cent ;
– stearic acid : 3.0 per cent to 19.0 per cent ;
– oleic acid and isomers : 54.0 per cent to 78.0 per cent ;
– linoleic acid and isomers : maximum 10.0 per cent ;
– arachidic acid : 1.0 per cent to 3.0 per cent ;
– eicosenoic acids : maximum 2.1 per cent ;
– behenic acid : 1.0 per cent to 5.0 per cent ;
– erucic acid and isomers : maximum 0.5 per cent ;
– lignoceric acid : 0.5 per cent to 3.0 per cent.
Nickel : maximum 1 ppm.
Atomic absorption spectrometry (2.2.23, Method II).
Test solution. Into a platinum or silica crucible previously tared after ignition introduce 5.0 g.
Cautiously heat and introduce into the substance a wick formed from twisted ashless filter paper.
Ignite the wick. When the substance has ignited stop heating. After combustion, ignite in a muffle
furnace at about 600 ± 50 °C. Continue ignition until white ash is obtained. After cooling, take up
the residue with 2 quantities, each of 2 mL, of dilute hydrochloric acid R and transfer into a 25 mL
graduated flask. Add 0.3 mL of nitric acid R and dilute to 25.0 mL with water R.
Reference solutions. Prepare 3 reference solutions by adding 1.0 mL, 2.0 mL and 4.0 mL of nickel
standard solution (0.2 ppm Ni) R to 2.0 mL of the test solution and diluting to 10.0 mL with water R.
Source : nickel hollow-cathode lamp.
Wavelength : 232 nm.
Atomisation device : graphite furnace.
Carrier gas : argon R.
STORAGE
Protected from light.
LABELLING
The label states the nominal drop point.


Identification : reference to the test for composition of fatty acids has been added since it is
more specific than the identification of fatty oils by TLC ; the latter has been maintained in the
second identification.
XXXX:0263
ARACHIS OIL, REFINED
Arachidis oleum raffinatum

DEFINITION
The refined fatty oil obtained from the shelled seeds of Arachis hypogaea L. A suitable antioxidant
may be added.
CHARACTERS
Appearance : clear, yellowish, viscous liquid.
Solubility : very slightly soluble in ethanol (96 per cent), miscible with light petroleum.
Relative density : about 0.915.
It solidifies at about 2 °C.
IDENTIFICATION
First identification : B.
Second identification : A.
A. Identification of fatty oils by thin-layer chromatography (2.3.2).
Results : the chromatogram obtained is similar to the corresponding chromatogram shown in
Figure 2.3.2.-1.
B. Composition of fatty acids (see Tests).
TESTS
Acid value (2.5.1) : maximum 0.5, determined on 10.0 g.
Peroxide value (2.5.5, Method A) : maximum 5.0.
Unsaponifiable matter (2.5.7) : maximum 1.0 per cent, determined on 5.0 g.
Alkaline impurities (2.4.19). It complies with the test.
Composition of fatty acids. (2.4.22, Method A). Use the mixture of calibrating substances in
Table 2.4.22.-3.
Composition of the fatty-acid fraction of the oil :
– saturated fatty acids of chain length less than C16 : maximum 0.4 per cent ;
– palmitic acid : 5.0 per cent to 14.0 per cent ;
– stearic acid : 1.3 per cent to 6.5 per cent ;
– oleic acid : 35.0 per cent to 76.0 per cent ;
– linoleic acid : 8.0 per cent to 43.0 per cent ;
– linolenic acid : maximum 0.6 per cent ;
– arachidic acid : 0.5 per cent to 3.0 per cent ;
– eicosenoic acid : 0.5 per cent to 3.0 per cent ;
– behenic acid : 1.0 per cent to 5.0 per cent ;
– erucic acid : maximum 0.5 per cent ;
– lignoceric acid : 0.5 per cent to 3.0 per cent.
Water (2.5.32) : maximum 0.1 per cent, determined on 1.00 g.

STORAGE
In a well-filled container, protected from light.

Reference: PA/PH/Exp. 13A/T (16) 58 ANP
Identification A : updated to allow better discrimination from other herbal drugs.
Sambucus nigra : test for adulteration with Sambucus nigra L. introduced.

XXXX:1588
BILBERRY FRUIT, DRIED
Myrtilli fructus siccus
DEFINITION
Dried ripe fruit of Vaccinium myrtillus L.
Content : minimum 1.0 per cent of tannins, expressed as pyrogallol (C6H6O3 ; Mr 126.1) (dried
drug).
IDENTIFICATION
A. Dried bilberry is a dark blackish-blue, subglobular, shrunken berry about 5 mm in diameter

covered in a whitish bloom, with a scar or, rarely, a fragment of the pedicel at the lower end.
The upper end is slightly depressed and surmounted by the remains of the style and of the
persistent calyx, which appears as a circular fold and the remains of the style. The deep violet,
fleshy mesocarp contains numerous (more than 10) small, brown, ovoid seeds.
B. Microscopic examination (2.8.23). The powder is violet-brown. Examine under a microscope

using chloral hydrate solution R. The powder shows : violet-pink sclereids from the endocarp
and the mesocarp, usually aggregated, with thick, channelled walls ; reddish-brown fragments
of the epicarp consisting of polygonal cells with moderately thickened walls ; brownish-yellow
fragments of the outer seed testa made up of elongated cells with U-shaped thickened walls ;
clusters and prisms crystals of various size of calcium oxalate.
B. Microscopic examination (2.8.23). The powder is violet-red. Examine under a microscope

using chloral hydrate solution R. The powder shows the following diagnostic characters (Figure
1588.-1) : fragments of dark red epicarp [A] consisting of small groups of 2-4 polygonal,
rectangular or quadrangular cells ; in each group, the inner walls are thin [Aa] whereas the
outer walls are regularly thickened [Ab] ; fragments of mesocarp [B] consisting of large,
rounded, thin-walled cells associated with fine spiral vessels [Ba] ; numerous sclereids,
isolated or in small groups, from the mesocarp [C] ; groups of sclereids from the endocarp [D] ;
yellowish-brown fragments from the testa, with large sclereids with walls that are extensively
channelled and pitted (surface view [E]) and with U-shaped thickenings (transverse section
[F]) ; irregular cluster crystals of calcium oxalate [G] and isolated prisms of calcium oxalate [H]
from the mesocarp.
PA/PH/Exp. 13A/T (16) 58 ANP

Figure 1588.-1. – Illustration for identification test B of powdered herbal drug of dried bilberry fruit
Test solution. To 2 g of the powdered herbal drug (355) (2.9.12) add 20 mL of methanol R.
Shake for 15 min and filter.
Reference solution. Dissolve 0.10 g of bilberry dry extract HRS in 25 mL of methanol R. Stir
for 15 min and filter.
Plate : TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel F254 plate R (2-10 µm)(6)].
Mobile phase : anhydrous formic acid R, water R, butanol R (16:19:65 V/V/V).
Application : 10 µL [or 2 µL] as bands of 15 mm [or 8 mm].
Development : over a path of 10 cm [or 6 cm].
Drying : in air.
Detection : examine in daylight.
reference solution and the test solution. Furthermore, other faint zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
_______ _______
A violet-red zone A violet-red zone

A violet zone A violet zone
A broad unresolved bluish-violet zone A broad unresolved bluish-violet zone
_______ _______
(6) Merck silica gel Si 60 F254 HPTLC glass plates are suitable.

TESTS
Sambucus nigra. The presence of glossy, violet-black, ovoid berries without the circular fold due
to the calyx, containing not more than 4 seeds indicates adulteration with Sambucus nigra L.
drug by drying in an oven at 105 °C for 2 h.
ASSAY
Tannins (2.8.14). Use 1.500 g of the powdered herbal drug (355) (2.9.12).

Identification A : updated to allow better discrimination from other herbal drugs.
Sambucus nigra : test for adulteration with Sambucus nigra L. introduced.

XXXX:1602
BILBERRY FRUIT, FRESH
Myrtilli fructus recens
DEFINITION
Fresh or frozen, ripe fruit of Vaccinium myrtillus L.
Content : minimum 0.30 per cent of anthocyanins, expressed as cyanidin 3-O-glucoside chloride
(chrysanthemin, C21H21ClO11 ; Mr 484.8) (dried drug).
IDENTIFICATION
A. The fresh fruit is a blackish-blue globular berry about 5 mm in diameter, covered in a whitish
bloom. Its lower end shows a scar or, rarely, a fragment of the pedicel. The upper end is
flattened slightly depressed and is surmounted by the remains of the persistent style and of the
persistent calyx, which appears as a circular fold. The violet, fleshy mesocarp includes 4 to 5
locules containing numerous (more than 10) small, brown, ovoid seeds.
B. The crushed fresh fruit is violet-red. Examine under a microscope using chloral hydrate
solution R. It shows violet-pink sclereids from the endocarp and the mesocarp, usually
aggregated, with thick, channelled walls ; reddish-brown fragments of the epicarp consisting
of polygonal cells with moderately thickened walls; brownish-yellow fragments of the outer
layer of the testa composed of elongated cells with U-shaped thickened walls ; cluster crystals
of calcium oxalate.
B. The crushed fresh fruit is violet-red. Examine small fragments under a microscope using
chloral hydrate solution R. They show the following diagnostic character (Figure 1602.-1) :
fragments of dark red epicarp [A] consisting of small groups of 2-4 polygonal, rectangular or
quadrangular cells ; in each group, the inner walls are thin [Aa] whereas the outer walls are
regularly thickened [Ab] ; fragments of mesocarp [B] consisting of large, rounded, thin-walled
cells associated with fine spiral vessels [Ba] ; numerous sclereids, isolated or in small groups,
from the mesocarp [C] ; groups of sclereids from the endocarp [D] ; yellowish-brown fragments
from the testa, with large sclereids with walls that are extensively channelled and pitted (surface
view [E]) and with U-shaped thickenings (transverse section [F]); irregular cluster crystals of
calcium oxalate [G] and isolated prisms of calcium oxalate [H] from the mesocarp.

Figure 1602.-1. – Illustration for identification test B of the herbal drug of fresh bilberry fruit
Test solution. To 5 g of the freshly crushed drug, add 20 mL of methanol R. Stir for 15 min
and filter.
Reference solution. Dissolve 0.10 g of bilberry dry extract HRS in 25 mL of methanol R. Stir
for 15 min and filter.
Plate : TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel F254 plate R (2-10 µm)(7)].
Mobile phase : anhydrous formic acid R, water R, butanol R (16:19:65 V/V/V).
Application : 10 µL [or 2 µL] as bands of 15 mm [or 8 mm].
Drying : in air.
Detection : examine in daylight.
reference solution and the test solution. Furthermore, other faint zones may be present in the
chromatogram obtained with the test solution.
Top of the plate
_______ _______
A violet-red zone A violet-red zone

A broad unresolved bluish-violet zone A broad unresolved bluish-violet zone
_______ _______
(7) Merck silica gel Si 60 F254 HPTLC glass plates are suitable.

TESTS
Sambucus nigra. The presence of glossy, violet-black, ovoid berries without the circular fold due
to the calyx, containing not more than 4 seeds indicates adulteration with Sambucus nigra L.
Loss on drying (2.2.32) : 80.0 per cent to 90.0 per cent, determined on 5.000 g of the freshly
crushed drug by drying in an oven at 105 °C.
ASSAY
Crush 50 g immediately before use. To about 5.00 g of the crushed, accurately weighed drug, add
95 mL of methanol R. Stir mechanically for 30 min. Filter into a 100.0 mL volumetric flask. Rinse
the filter and dilute to 100.0 mL with methanol R. Prepare a 50-fold dilution of this solution in a
0.1 per cent V/V solution of hydrochloric acid R in methanol R.
Measure the absorbance (2.2.25) of the solution at 528 nm, using a 0.1 per cent V/V solution of
hydrochloric acid R in methanol R as the compensation liquid.
Calculate the percentage content of anthocyanins, expressed as cyanidin 3-O-glucoside chloride,
using the following expression :
718 = specific absorbance of cyanidin 3-O-glucoside chloride at 528 nm ;

A = absorbance at 528 nm ;
m = mass of the substance to be examined in grams.
STORAGE
When frozen, store at or below − 18 °C.


Identification : IR identification introduced to replace former identification tests A, B and C.
Related substances : test added to control 3 new impurities.
Assay : end-point determination by colour indicator replaced by potentiometry, thereby also
avoiding the use of dibutyl phthalate (REACH).
Impurities : section describing impurities introduced.
XXXX:0382
CHLOROBUTANOL
Chlorobutanolum
C4H7Cl3O Mr 177.5
[57-15-8]
DEFINITION
1,1,1-Trichloro-2-methylpropan-2-ol.
Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or colourless crystals, sublimes readily.
Solubility : slightly soluble in water, very soluble in ethanol (96 per cent), soluble in glycerol (85 per
cent).
mp : about 95 °C (without previous drying).
IDENTIFICATION
A. Add about 20 mg to a mixture of 1 mL of pyridine R and 2 mL of strong sodium hydroxide
solution R. Heat in a water-bath and shake. Allow to stand. The pyridine layer becomes red.
Comparison : chlorobutanol CRS.
B. Add about 20 mg to 5 mL of ammoniacal silver nitrate solution R and warm slightly. A black
precipitate is formed.
C. To about 20 mg add 3 mL of 1 M sodium hydroxide and shake to dissolve. Add 5 mL of water R
and then, slowly, 2 mL of iodinated potassium iodide solution R. A yellowish precipitate is
formed.
D. B. Water (see Tests).
TESTS
Solution S. Dissolve 5 g in ethanol (96 per cent) R and dilute to 10 mL with the same solvent.
Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than reference solution BY5 (2.2.2, Method II).
Acidity. To 4 mL of solution S add 15 mL of ethanol (96 per cent) R and 0.1 mL of bromothymol
blue solution R1. Not more than 1.0 mL of 0.01 M sodium hydroxide is required to change the
colour of the indicator to blue.

Related substances. Head-space gas chromatography (2.2.28).
Pharmacopoeia.
1. impurity B 3. impurity A
2. impurity C 4. dimethylformamide
Figure 0382.-1. – Chromatogram for the test for related substances of chlorobutanol : solvent
mixture spiked with impurities A, B and C.
Solvent mixture : 60 per cent V/V solution of dimethylformamide R.
Test solution. Introduce 2.000 g of the substance to be examined into a vial, add 5.0 mL of the
solvent mixture and close the vial immediately.
Reference solution (a). Dissolve 60.0 mg of chloroform R (impurity A) in the solvent mixture and
dilute to 50.0 mL with the solvent mixture. Shake well.
Reference solution (b). Dissolve 500.0 mg of acetone R (impurity B) in the solvent mixture and
Reference solution (c). Dissolve 50.0 mg of 2-propanol R (impurity C) in the solvent mixture and
Reference solution (d). Dilute a mixture of 4.0 mL of reference solution (a), 8.0 mL of reference
solution (b) and 8.0 mL of reference solution (c) to 200.0 mL with the solvent mixture.
Column :
– material: fused silica ;
– size : l = 30 m, Ø = 0.53 mm ;
– stationary phase : poly[(cyanopropyl)(phenyl)][dimethyl]siloxane R (film thickness 3 µm)(8).
Carrier gas : nitrogen R.
Split ratio : 1:3.
Static head-space conditions that may be used :
– equilibration temperature : 90 °C ;
– equilibration time : 20 min ;
– pressurisation time : 30 s ;
– incubation time : 50 min.
Temperature :
(8) DB-624 is suitable.

Time Temperature
(min) (°C)
Column 0 - 25 40 → 220
25 - 30 220
Injection port 220
Detector 230

Injection : 1.0 mL.
Run time : 3 times the retention time of impurity B.
Identification of impurities : use the chromatogram obtained with reference solution (a) to identify
the peak due to impurity A ; use the chromatogram obtained with reference solution (b) to identify
the peak due to impurity B ; use the chromatogram obtained with reference solution (c) to identify
the peak due to impurity C.
Relative retention with reference to impurity B (retention time = about 14 min) : impurity C = about
1.1 ; impurity A = about 2.0.
System suitability : reference solution (d) :
– resolution : minimum 1.5 between the peaks due to impurities B and C.
Calculation of contents :
– for impurity A, use the concentration of impurity A in reference solution (d) ;
– for impurities other than A, use the concentration of impurity B in reference solution (d).
Limits :
– impurity A : maximum 60 ppm ;
– unspecified impurities : for each impurity, maximum 0.10 per cent ;
– total : maximum 0.15 per cent ;
– reporting threshold : 0.05 per cent, except for impurity A.
Chlorides (2.4.4) : maximum 300 ppm.
Dissolve 0.17 g in 5 mL of ethanol (96 per cent) R and dilute to 15 mL with water R. When
preparing the standard, replace the 5 mL of water R by 5 mL of ethanol (96 per cent) R.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g.
ASSAY
Dissolve 0.100 g in 20 mL of ethanol (96 per cent) R. Add 10 mL of dilute sodium hydroxide
solution R, heat in a water-bath for 5 min and cool. Add 20 mL of dilute nitric acid R, 25.0 mL of
0.1 M silver nitrate and 2 mL of dibutyl phthalate R and shake vigorously. Add 2 mL of ferric
ammonium sulfate solution R2 and titrate with 0.1 M ammonium thiocyanate until an orange
colour is obtained.
Dissolve 0.050 g in 20 mL of ethanol (96 per cent) R in a 50 mL centrifuge tube with cap. Add
10 mL of dilute sodium hydroxide solution R, close tightly and heat in a water-bath for 10 min. Cool
and transfer quantitatively to a titration vessel using 100 mL of water R. Add 20 mL of dilute nitric
acid R and titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M silver nitrate is equivalent to 5.92 mg of C4H7Cl3O.
STORAGE
In an airtight container.
IMPURITIES
Specified impurities : A.
Other detectable impurities (the following substances would, if present at a sufficient level,
be detected by one or other of the tests in the monograph. They are limited by the general
acceptance criterion for other/unspecified impurities and/or by the general monograph

Substances for pharmaceutical use (2034). It is therefore not necessary to identify these
impurities for demonstration of compliance. See also 5.10. Control of impurities in substances for
pharmaceutical use) : B, C.
A. trichloromethane (chloroform),
B. propan-2-one (acetone),
C. propan-2-ol (isopropyl alcohol).


Identification : IR identification introduced to replace former identification tests A, B and C.
Related substances : test added to control 3 new impurities.
Assay : end-point determination by colour indicator replaced by potentiometry, thereby also
avoiding the use of dibutyl phthalate (REACH).
Impurities : section describing impurities introduced.
XXXX:0383
CHLOROBUTANOL HEMIHYDRATE
Chlorobutanolum hemihydricum
C4H7Cl3O,½H2O Mr 186.5
[6001-64-5]
DEFINITION
1,1,1-Trichloro-2-methylpropan-2-ol hemihydrate.
Content : 98.0 per cent to 101.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or colourless crystals, sublimes readily.
Solubility : slightly soluble in water, very soluble in ethanol (96 per cent), soluble in glycerol (85 per
cent).
mp : about 78 °C (without previous drying).
IDENTIFICATION
A. Add about 20 mg to a mixture of 1 mL of pyridine R and 2 mL of strong sodium hydroxide
solution R. Heat in a water-bath and shake. Allow to stand. The pyridine layer becomes red.
Comparison : chlorobutanol hemihydrate CRS.
B. Add about 20 mg to 5 mL of ammoniacal silver nitrate solution R and warm slightly. A black
precipitate is formed.
C. To about 20 mg add 3 mL of 1 M sodium hydroxide and shake to dissolve. Add 5 mL of water R
and then, slowly, 2 mL of iodinated potassium iodide solution R. A yellowish precipitate is
formed.
D. B. Water (see Tests).
TESTS
Solution S. Dissolve 5 g in ethanol (96 per cent) R and dilute to 10 mL with the same solvent.
Appearance of solution. Solution S is not more opalescent than reference suspension II (2.2.1)
and not more intensely coloured than reference solution BY5 (2.2.2, Method II).
Acidity. To 4 mL of solution S add 15 mL of ethanol (96 per cent) R and 0.1 mL of bromothymol
blue solution R1. Not more than 1.0 mL of 0.01 M sodium hydroxide is required to change the
colour of the indicator to blue.

Related substances. Head-space gas chromatography (2.2.28).
Pharmacopoeia.
1. impurity B 3. impurity A
2. impurity C 4. dimethylformamide
Figure 0383.-1. – Chromatogram for the test for related substances of chlorobutanol hemihydrate :
solvent mixture spiked with impurities A, B and C.
Solvent mixture : 60 per cent V/V solution of dimethylformamide R.
Test solution. Introduce 2.000 g of the substance to be examined into a vial, add 5.0 mL of the
solvent mixture and close the vial immediately.
Reference solution (a). Dissolve 60.0 mg of chloroform R (impurity A) in the solvent mixture and
Reference solution (b). Dissolve 500.0 mg of acetone R (impurity B) in the solvent mixture and
Reference solution (c). Dissolve 50.0 mg of 2-propanol R (impurity C) in the solvent mixture and
Reference solution (d). Dilute a mixture of 4.0 mL of reference solution (a), 8.0 mL of reference
solution (b) and 8.0 mL of reference solution (c) to 200.0 mL with the solvent mixture.
Column :
– material: fused silica ;
– size : l = 30 m, Ø = 0.53 mm ;
Carrier gas : nitrogen R.
Split ratio : 1:3.
– pressurisation time : 30 s.
– incubation time : 50 min.
Temperature :
(9) DB-624 is suitable.

Time Temperature
(min) (°C)
Column 0 - 25 40 → 220
25 - 30 220
Injection port 220
Detector 230

Injection : 1.0 mL.
Run time : 3 times the retention time of impurity B.
the peak due to impurity A ; use the chromatogram obtained with reference solution (b) to identify
the peak due to impurity B ; use the chromatogram obtained with reference solution (c) to identify
the peak due to impurity C.
Relative retention with reference to impurity B (retention time = about 14 min) : impurity C = about
1.1 ; impurity A = about 2.0.
System suitability : reference solution (d) :
– resolution : minimum 1.5 between the peaks due to impurities B and C.
Calculation of contents :
– for impurity A, use the concentration of impurity A in reference solution (d) ;
– for impurities other than A, use the concentration of impurity B in reference solution (d).
Limits :
– impurity A : maximum 60 ppm ;
– reporting threshold : 0.05 per cent, except for impurity A.
To 1 mL of solution S add 4 mL of ethanol (96 per cent) R and dilute to 15 mL with water R. When
preparing the standard, replace the 5 mL of water R by 5 mL of ethanol (96 per cent) R.
Water (2.5.12) : 4.5 per cent to 5.5 per cent, determined on 0.300 g.
ASSAY
Dissolve 0.100 g in 20 mL of ethanol (96 per cent) R. Add 10 mL of dilute sodium hydroxide
solution R, heat in a water-bath for 5 min and cool. Add 20 mL of dilute nitric acid R, 25.0 mL of
0.1 M silver nitrate and 2 mL of dibutyl phthalate R and shake vigorously. Add 2 mL of ferric
ammonium sulfate solution R2 and titrate with 0.1 M ammonium thiocyanate until an orange
colour is obtained.
Dissolve 0.050 g in 20 mL of ethanol (96 per cent) R in a 50 mL centrifuge tube with cap. Add
10 mL of dilute sodium hydroxide solution R, close tightly and heat in a water-bath for 10 min. Cool
and transfer quantitatively to a titration vessel using 100 mL of water R. Add 20 mL of dilute nitric
acid R and titrate with 0.1 M silver nitrate, determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M silver nitrate is equivalent to 5.92 mg of C4H7Cl3O.
STORAGE
IMPURITIES
Specified impurities : A.

pharmaceutical use) : B, C.
A. trichloromethane (chloroform),
B. propan-2-one (acetone),
C. propan-2-ol (isopropyl alcohol).

Reference: PA/PH/Exp. 11/T (16) 62 ANP

Identification : it is proposed to delete the 2nd identification series,since the substance is not
used in pharmacies.
Related substances : TLC has been replaced by LC and the specifications have been updated
based on recent batch data.
XXXX:1088
CINCHOCAINE HYDROCHLORIDE
Cinchocaini hydrochloridum
C20H30ClN3O2 Mr 379.9
[61-12-1]
DEFINITION
2-Butoxy-N-[2-(diethylamino)ethyl]quinoline-4-carboxamide hydrochloride.
Content : 98.5 per cent to 101.0 per cent (dried substance).
CHARACTERS
Appearance : hygroscopic, very easily agglomerating, white or almost white, crystalline powder or
colourless crystals.
Solubility : very soluble in water, freely soluble in acetone, in ethanol (96 per cent) and in
methylene chloride.
IDENTIFICATION
First identification : B, E.
Second identification : A, C, D, E.
A. Dissolve 60.0 mg in 1 M hydrochloric acid and dilute to 100 mL with the same acid. Dilute 2 mL
of the solution to 100 mL with 1 M hydrochloric acid. Examined between 220 nm and 350 nm
(2.2.25), the solution shows two absorption maxima, at 246 nm and 319 nm. The ratio of the
absorbance measured at 246 nm to that measured at 319 nm is 2.7 to 3.0.
A. B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs of potassium chloride R.
Comparison : cinchocaine hydrochloride CRS.
C. Examine the chromatograms obtained in the test for related substances. The principal spot in
the chromatogram obtained with test solution (b) is similar in position and size to the principal
spot in the chromatogram obtained with reference solution (a).
D. Dissolve 0.5 g in 5 mL of water R. Add 1 mL of dilute ammonia R2. A white precipitate is
formed. Filter, wash the precipitate with five quantities, each of 10 mL, of water R and dry in a
desiccator. It melts at 64 °C to 66 °C (2.2.14).
B. E. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 5.0 g in carbon dioxide-free water R prepared from distilled water R and
dilute to 50 mL with the same solvent.
Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than
reference solution Y6 (2.2.2, Method II).
PA/PH/Exp. 11/T (16) 62 ANP

pH (2.2.3) : 5.0 to 6.0.

Dilute 10 mL of solution S to 50 mL with carbon dioxide-free water R.
Related substances. Examine by thin-layer chromatography (2.2.27), using as the coating
substance a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm.
Test solution (a). Dissolve 0.20 g of the substance to be examined in methanol R and dilute to
Test solution (b). Dilute 1 mL of test solution (a) to 10 mL with methanol R.
Reference solution (a). Dissolve 20 mg of cinchocaine hydrochloride CRS in methanol R and
Reference solution (b). Dilute 1 mL of test solution (b) to 20 mL with methanol R.
Reference solution (c). Dilute 1 mL of test solution (b) to 50 mL with methanol R.
Reference solution (d). Dissolve 20 mg of benzocaine CRS in methanol R and dilute to 5 mL
with the same solvent. Dilute 1 mL of the solution and 1 mL of reference solution (a) to 20 mL
with methanol R.
Apply separately to the plate 5 µL of each solution. Develop over a path of 15 cm using a mixture
of 1 volume of ammonia R, 5 volumes of methanol R, 30 volumes of acetone R and 50 volumes of
toluene R. Dry the plate in a current of warm air for 15 min. Examine in ultraviolet light at 254 nm.
Any spot in the chromatogram obtained with test solution (a), apart from the principal spot, is not
more intense than the principal spot in the chromatogram obtained with reference solution (b)
(0.5 per cent) and at most one such spot is more intense than the spot in the chromatogram
obtained with reference solution (c) (0.2 per cent). The test is not valid unless the chromatogram
obtained with reference solution (d) shows two clearly separated spots.
Related substances. Liquid chromatography (2.2.29).
Solvent mixture. Mobile phase B, mobile phase A (10:90 V/V).
Test solution. Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute
to 50.0 mL with the solvent mixture.
Reference solution (a). Dilute 1.0 mL of the test solution to 10.0 mL with the solvent mixture.
Dilute 1.0 mL of this solution to 100.0 mL with the solvent mixture.
Reference solution (b). Dissolve 2 mg of cinchocaine impurity A CRS and 2 mg of cinchocaine
impurity B CRS in the solvent mixture and dilute to 100.0 mL with the solvent mixture. Dilute
1.0 mL of the solution to 10.0 mL with the solvent mixture.
Column :
– size : l = 0.25 m, Ø = 3 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for chromatography R (5 µm)(10) ;
– temperature : 40 °C.
Mobile phase :
– mobile phase A : to 950 mL of water for chromatography R add 0.6 mL of phosphoric
acid R, adjust to pH 2.0 with phosphoric acid R and dilute to 1000.0 mL with water for
chromatography R ;
– mobile phase B : acetonitrile R1 ;
Time(11) Mobile phase A Mobile phase B
(min) (per cent V/V) (per cent V/V)
0-2 90 10
2 - 5.5 90 → 82 10 → 18
5.5 - 10.5 82 → 50 18 → 50
10.5 - 16.5 50 → 49 50 → 51
16.5 - 19.5 49 → 46 51 → 54
19.5 - 24.5 46 → 30 54 → 70
(10) Nucleodur C18 ISIS (Macherey-Nagel) is suitable.

(11) D0 (dwell volume used for development of the method) = 1.4 mL.


Injection : 10 µL.
Relative retention with reference to cinchocaine (retention time = about 10.5 min) :
impurity B = about 0.5 ; impurity A = about 0.6.
System suitability : reference solution (b) :
– resolution : minimum 3.0 between the peaks due to impurities A and B.
Calculation of percentage contents :
– for each impurity, use the concentration of cinchocaine in reference solution (a).
Limits :
– reporting threshold : 0.05 per cent.
Pharmacopoeia.
1. impurity C 3. impurity A 5. impurity D

2. impurity B 4. cinchocaine
Figure 1088.-1. – Chromatogram for the test for related substances : cinchocaine hydrochloride
spiked with impurities A to D
at 60 °C.
ASSAY
Dissolve 0.300 g in a mixture of 15.0 mL of 0.01 M hydrochloric acid and 50 mL of ethanol (96 per
cent) R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read
the volume added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 37.99 mg of C20H30ClN3O2.
STORAGE
In an airtight container, protected from light.
IMPURITIES
impurities for demonstration of compliance. See also 5.10. Control of impurities in substances
for pharmaceutical use) : A, B, C, D.

A. 2-chloro-N-[2-(diethylamino)ethyl]quinoline-4-carboxamide,
B. 2-hydroxyquinoline-4-carboxylic acid,
C. N-[2-(diethylamino)ethyl]-2-hydroxyquinoline-4-carboxamide,
D. 2-butoxyquinoline-4-carboxylic acid.

Reference: PA/PH/Exp. HOM/T (13) 21 ANP
XXXX:2705
DIGITALIS FOR HOMOEOPATHIC PREPARATIONS(12)
Digitalis purpurea ad praeparationes homoeopathicas

DEFINITION
Fresh leaf of Digitalis purpurea L, collected just before or during flowering.
IDENTIFICATION
A. The leaf is variable in size, usually 10-50 cm long and 4-15 cm wide, simple, entire, lanceolate,
oblong, and ending in a subacute apex. The margins of the lamina are crenate or dentate. It is
thick with a velvety or rough texture. The lamina is decurrent, attenuated along the midrib, the
whole forming a winged, triangular petiole, with purple-pink spots at the base. The venation is
pinnate, with the lateral veins leaving the midrib at about 45° ; they anastomose near the leaf
margin, forming arcs, and are connected to each other by an extensive network of tertiary
veinlets. The upper surface is greyish-green and pubescent, but sometimes almost glabrous.
The veins are sunken, forming depressed lines around bulging areas in the lamina. The lower
surface is paler and very tomentose ; the whitish veins are prominent giving the surface a
honeycomb appearance.
B. Take a fragment of the lower epidermis of the leaf. Examine under a microscope using chloral
hydrate solution R. The leaf has a smooth cuticle and shows epidermal cells up to 30-75 µm
long, with distinctly sinuous anticlinal walls, numerous anomocytic stomata (2.8.3) and 3
types of trichomes : covering trichomes often articulated and bent at right angles, uniseriate,
usually with 3-5 cells, sometimes collapsed, and a terminal cell covered in a sometimes
smooth but usually verrucose or slightly striated cuticle ; glandular trichomes with unicellular
stalks and globular bicellular heads ; glandular trichomes with multicellular, uniseriate stalks
and unicellular heads.
TESTS
Foreign matter (2.8.2) : maximum 5 per cent.
Loss on drying (2.2.32) : minimum 70.0 per cent, determined on 5.0 g of the comminuted herbal
Digitalis lanata. The presence of oval lanceolate, narrow leaves, with entire margins or with
dentate margins only near the apex, the existence of a few glandular trichomes of epidermal cells
with very characteristic beaded walls, and the absence of covering trichomes indicate adulteration
with Digitalis lanata Ehrh.
Digitalis lutea. The presence of sessile, lanceolate, denticulate, almost glabrous leaves and the
scarcity of smooth covering trichomes indicate adulteration with Digitalis lutea L.
Digitalis grandiflora. The presence of oblong to oval leaves with serrate margins, non-reticulate
venation and veins bearing rare, very large covering trichomes with large pits indicates
adulteration with Digitalis grandiflora All.
Mother tincture
The mother tincture complies with the requirements of the general monograph Mother tinctures
for homoeopathic preparations (2029).
DEFINITION
The mother tincture is prepared from the fresh leaf, collected just before or during flowering, of
Digitalis purpurea L.
(12) FRENCH TITLE : Digitalis purpurea pour préparations homéopathiques
PA/PH/Exp. HOM/T (13) 21 ANP

Content : 0.003 per cent m/m to 0.013 per cent m/m of digitoxin (C41H64O13 ; Mr 765).
PRODUCTION
The mother tincture is prepared from the comminuted herbal drug according to the following
methods prescribed in the monograph Methods of preparation of homoeopathic stocks and
potentisation (2371) :
– method 1.1.3 ;
– method 1.1.10, using ethanol (65 per cent V/V) and a maceration time of 3-5 weeks.
CHARACTERS
Appearance : light greenish-brown to brown liquid.
IDENTIFICATION
Test solution. Evaporate 10 mL of the mother tincture to be examined to dryness under reduced
pressure. Take up the residue with 1 mL of a mixture of equal volumes of ethyl acetate R and
methanol R.
Reference solution. Dissolve 5 mg of digitoxin R and 2 mg of gitoxin R in a mixture of equal
volumes of ethyl acetate R and methanol R and dilute to 10 mL with the same mixture of solvents.
Plate : TLC silica gel G plate R (5-40 µm) or [TLC silica gel plate R (2-10 µm)].
Mobile phase : water R, methanol R, ethyl acetate R (7.5:10:75 V/V/V).
Application : 20 µL [or 10 µL] as bands.
Drying : in air.
Detection : treat with a mixture of 2 volumes of a 10 g/L solution of chloramine R and 8 volumes of
a 250 g/L solution of trichloroacetic acid R in ethanol (96 per cent) R, then heat at 100-105 °C for
10 min ; examine in ultraviolet light at 365 nm.
Results : see below the sequence of fluorescent zones present in the chromatograms obtained
with the reference solution and the test solution. Furthermore, other faint zones may be present in
the chromatogram obtained with the test solution.
Top of the plate
_______ _______
Digitoxin : a bluish-green zone A bluish-green zone (digitoxin)

Gitoxin : a light blue zone A light blue zone (gitoxin)
_______ _______
A faint brownish-yellow zone may be present

A faint light blue zone may be present
TESTS
Relative density (2.2.5) : 0.935 to 0.955 (method 1.1.3).
Ethanol (2.9.10) : 60 per cent V/V to 70 per cent V/V (method 1.1.10).
Dry residue (2.8.16) : minimum 2.5 per cent.
ASSAY
Test solution. Dilute 8.0 g of the mother tincture to be examined to 10.0 mL with water for
chromatography R and filter through a membrane filter (nominal pore size 0.45 µm).
Reference solution (a). Dissolve 10.0 mg of digitoxin CRS in methanol R2 and dilute to 10.0 mL
with the same solvent. Dilute 2.00 mL of the solution to 20.0 mL with a mixture of equal volumes
of methanol R2 and water for chromatography R.

Reference solution (b). Dissolve 5.0 mg of lanatoside C R and 5.0 mg of digoxin CRS in
methanol R2 and dilute to 10.0 mL with the same solvent. Dilute 1.0 mL of the solution to 20.0 mL
with a mixture of equal volumes of methanol R2 and water for chromatography R.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : octadecylsilyl silica gel for chromatography R (3 µm)(13) ;
Mobile phase :
– mobile phase A : water for chromatography R ;
– mobile phase B : acetonitrile for chromatography R ;

0-2 80 20
2 - 32 80 → 40 20 → 60
32 - 33 40 → 80 60 → 20
33 - 38 80 20
Injection : 10 µL.
Retention time : digitoxin = about 24 min.
– resolution : minimum 1.5 between the peaks due to lanatoside C and digoxin.
Calculate the percentage content of digitoxin using the following expression :
A1 = area of the peak due to digitoxin in the chromatogram obtained with the test
solution ;
A2 = area of the peak due to digitoxin in the chromatogram obtained with reference
solution (a) ;
m1 = mass of the mother tincture to be examined used to prepare the test solution,
in grams ;
m2 = mass of digitoxin CRS used to prepare reference solution (a), in grams ;
p = percentage content of digitoxin in digitoxin CRS.
(13) Luna C18 is suitable.


Pharmacopoeia.
1. digitoxin
Figure 2705.-1. – Chromatogram for the assay of the mother tincture of Digitalis for homoeopathic
preparations : test solution
Pharmacopoeia.
1. lanatoside C 2. digoxin
Figure 2705.-2. – Chromatogram for the assay of the mother tincture of Digitalis for homoeopathic
preparations : reference solution (b)

Reference: PA/PH/Exp. VIT/T (16) 1 ANP

Related substances and assay : a new method allowing baseline separation of the peaks due to
impurity A, pre-ergocalciferol and ergocalciferol is proposed ; limits updated based on available
data.
XXXX:0082
ERGOCALCIFEROL
Ergocalciferolum
C28H44O Mr 396.7
[50-14-6]
DEFINITION
(5Z,7E,22E)-9,10-Secoergosta-5,7,10(19),22-tetraen-3β-ol.
Content : 96.5 97.0 per cent to 102.0 per cent.
A suitable antioxidant may be added.
A reversible isomerisation to pre-ergocalciferol takes place in solution, depending on temperature
and time. The activity is due to both compounds (see Assay).
1 mg of ergocalciferol is equivalent to 40 000 IU of antirachitic activity (vitamin D) in rats.
CHARACTERS
Appearance : white or slightly yellowish, crystalline powder or white or almost white crystals.
Solubility : practically insoluble in water, freely soluble in ethanol (96 per cent) and in methanol,
soluble in fatty oils.
It is sensitive to air, heat and light. Solutions in volatile solvents are unstable and are to be used
immediately.
IDENTIFICATION
Infrared absorption spectrophotometry (2.2.24).
Comparison : ergocalciferol CRS.
TESTS
Specific optical rotation (2.2.7) : + 103 to + 107.
Dissolve 0.200 g rapidly and without heating in aldehyde-free alcohol R and dilute to 25.0 mL with
the same solvent. Examine within 30 min of preparing the solution.
Reducing substances. Dissolve 0.1 g in aldehyde-free alcohol R and dilute to 10.0 mL with the
same solvent. Add 0.5 mL of a 5 g/L solution of tetrazolium blue R in aldehyde-free alcohol R
and 0.5 mL of dilute tetramethylammonium hydroxide solution R. Allow to stand for exactly
5 min and add 1.0 mL of glacial acetic acid R. Prepare a reference solution at the same time
and in the same manner using 10.0 mL of a solution containing 0.2 µg/mL of hydroquinone R in
aldehyde-free alcohol R. Measure the absorbance (2.2.25) of the 2 solutions at 525 nm using as
the compensation liquid 10.0 mL of aldehyde-free alcohol R treated in the same manner. The
absorbance of the test solution is not greater than that of the reference solution (20 ppm).
PA/PH/Exp. VIT/T (16) 1 ANP

Impurity B. Liquid chromatography (2.2.29). Carry out the test as rapidly as possible, avoiding
exposure to actinic light and air.
Test solution. Dissolve 25.0 mg of the substance to be examined without heating in methanol R
and dilute to 25.0 mL with the same solvent.
Reference solution. Dissolve 5.0 mg of ergosterol CRS (impurity B) without heating in methanol R
and dilute to 50.0 mL with the same solvent. Dilute 1.0 mL of the solution to 50.0 mL with
methanol R.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
Mobile phase : methanol R.
Injection : 20 µL.
Run time : 2.5 times the retention time of ergocalciferol.
Relative retention with reference to ergocalciferol (retention time = about 7 min) : impurity B = about
1.6.
Limit :
– impurity B : not more than the area of the principal peak in the chromatogram obtained with the
reference solution (0.2 per cent).
Pharmacopoeia.
1. ergocalciferol 2. impurity B
Figure 0082.-1. – Chromatogram for the test for impurity B of ergocalciferol: test solution
Related substances. Liquid chromatography (2.2.29). Carry out the test as rapidly as possible,
avoiding exposure to actinic light and air.
Test solution. Dissolve 25.0 mg of the substance to be examined without heating in methanol R
Reference solution (a). Dissolve 5 mg of ergocalciferol for system suitability CRS (containing
impurities A, F and G) in methanol R and dilute to 5.0 mL with the same solvent. Heat in a
water-bath at 90 °C under a reflux condenser for 45 min and allow to cool.
(15) Nucleosil 100-5 C18 HD is suitable.

Reference solution (b). Dissolve 25.0 mg of ergocalciferol CRS without heating in methanol R
Reference solution (c). Dilute 1.0 mL of the test solution to 100.0 mL with methanol R.
Reference solution (d). Dilute 5.0 mL of reference solution (c) to 50.0 mL with methanol R.
Test solution. Dissolve 15.0 mg of the substance to be examined in the mobile phase and dilute to
25.0 mL with the mobile phase. Dilute 1.0 mL of the solution to 10.0 mL with the mobile phase.
Reference solution (a). Dissolve 5.0 mg of ergocalciferol for system suitability CRS (containing
impurities A, F and G) in 10 mL of the mobile phase. Heat in a water-bath at 90 °C under a
reflux-condenser for 45 min and allow to cool (in-situ degradation to obtain pre-ergocalciferol).
Dilute 3.0 mL of the solution to 25.0 mL with the mobile phase.
Reference solution (b). Dissolve 15.0 mg of ergocalciferol CRS in the mobile phase and dilute to
Reference solution (c). Dilute 1.0 mL of the test solution to 100.0 mL with the mobile phase.
Reference solution (d). Dilute 1.0 mL of reference solution (c) to 10.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : end-capped octadecylsilyl silica gel for chromatography R base-deactivated
end-capped octadecylsilyl silica gel for chromatography R (5 µm)(16) ;
Mobile phase : water for chromatography R, methanol R (5:95 V/V) methanol R, acetonitrile R
(10:90 V/V).
Flow rate : 1.5 1.0 mL/min.
Detection : spectrophotometer at 254 265 nm.
Injection : 20 µL of the test solution and reference solutions (a), (c) and (d).
Run time : twice the retention time of ergocalciferol.
Identification of impurities : use the chromatogram supplied with ergocalciferol for system
suitability CRS and the chromatogram obtained with reference solution (a) to identify the peaks
due to impurities A, F and G, and pre-ergocalciferol.
Relative retention with reference to ergocalciferol (retention time = about 16 15 min) :
impurity F = about 0.7 0.6 ; impurity A = about 0.9 0.8 ; pre-ergocalciferol = about 0.95 0.9 ;
impurity G = about 1.2.
System suitability : reference solution (a) :
– resolution : minimum 4.2 between the peaks due to ergocalciferol and impurity G ; minimum 2.0
between the peaks due to impurity A and pre-ergocalciferol ; minimum 2.5 between the peaks
due to pre-ergocalciferol and ergocalciferol.
– peak-to-valley ratio : minimum 1.2, where Hp = height above the baseline of the peak due to
impurity A and Hv = height above the baseline of the lowest point of the curve separating this
peak from the peak due to pre-ergocalciferol ; minimum 1.5, where Hp = height above the
baseline of the peak due to pre-ergocalciferol and Hv = height above the baseline of the lowest
point of the curve separating this peak from the peak due to ergocalciferol.
– for impurities A, F and G, use the concentration of ergocalciferol in reference solution (c) ;
– for impurities other than A, F and G, use the concentration of ergocalciferol in reference
solution (d).
Limits :
– impurity G : not more than 1.5 times the area of the principal peak in the chromatogram obtained
with reference solution (c) (1.5 per cent) maximum 1.5 per cent ;
– impurities A, F : for each impurity, not more than 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (c) (0.5 per cent) maximum 0.5 per cent ;
(16) Hypersil BDS C18 is suitable.

– unspecified impurities : for each impurity, not more than the area of the principal peak in the
chromatogram obtained with reference solution (d) (0.10 per cent) maximum 0.10 per cent ;
– total : maximum 2.5 2.0 per cent ;
– disregard limitreporting threshold : 0.5 times the area of the principal peak in the
chromatogram obtained with reference solution (d) (0.05 per cent) ; disregard any peak due to
pre-ergocalciferol or the antioxidant.
Pharmacopoeia.
1. impurity F 3. pre-ergocalciferol 5. impurity G
2. impurity A 4. ergocalciferol
Figure 0082.-2. – Chromatogram for the test for related substances of ergocalciferol: test solution
spiked with impurities A, F and G
ASSAY
Carry out the assay as rapidly as possible, avoiding exposure to actinic light and air.
Liquid chromatography (2.2.29) as described in the test for related substances with the following
modification.
Injection : test solution and reference solution (b).
Calculate the percentage content of C28H44O taking into account the assigned content of
ergocalciferol CRS, and if necessary present, the peak due to pre-ergocalciferol in the test
solution.
STORAGE
Under an inert gas, in an airtight container, protected from light, at a temperature of 2 °C to 8 °C.
The contents of an opened container are to be used immediately.
LABELLING
The label states the name and concentration of any added antioxidant.
IMPURITIES
Specified impurities : A, B, F, G.
pharmaceutical use) : C, D, E.

A. (5E,7E,22E)-9,10-secoergosta-5,7,10(19),22-tetraen-3β-ol (trans-vitamin D2),
B. (22E)-ergosta-5,7,22-trien-3β-ol (ergosterol),
C. (22E)-9β,10α-ergosta-5,7,22-trien-3β-ol (lumisterol2),
D. (6E,22E)-9,10-secoergosta-5(10),6,8(14),22-tetraen-3β-ol (iso-tachysterol2),
E. (6E,22E)-9,10-secoergosta-5(10),6,8,22-tetraen-3β-ol (tachysterol2),
F. (5Z,7E,22E)-9,10-secoergosta-5,7,10(19),22,24(241)-pentaen-3β-ol,

G. (5Z,7E)-9,10-secoergosta-5,7,10(19)-trien-3β-ol (vitamin D4).


Dimers and related substances of higher molecular mass : test renamed ‘Dimers and related
substances with molecular masses greater than that of erythropoietin’; erythropoietin for SEC
system suitability CRS and new requirements for chromatographic column introduced.
XXXX:1316
ERYTHROPOIETIN CONCENTRATED SOLUTION
Erythropoietini solutio concentrata
Mr approx. 30 600
DEFINITION
Solution containing a family of closely-related glycoproteins which are indistinguishable from the
naturally occurring human erythropoietin (urinary erythropoietin) in terms of amino acid sequence
(165 amino acids) and average glycosylation pattern, at a concentration of 0.5-10 mg/mL. It
may also contain buffer salts and other excipients. It has a potency of not less than 100 000 IU
per milligram of active substance determined using the conditions described under Assay and
in the test for protein.
PRODUCTION
Erythropoietin is produced in rodent cells in vitro by a method based on recombinant DNA
technology.
Prior to batch release, the following tests are carried out on each batch of the final product, unless
exemption has been granted by the competent authority.
Host cell-derived proteins : the limit is approved by the competent authority.
Host cell- and vector-derived DNA : the limit is approved by the competent authority.
CHARACTERS
Appearance : clear or slightly turbid, colourless solution.
IDENTIFICATION
A. It gives the appropriate response when examined using the conditions described under Assay.
B. Capillary zone electrophoresis (2.2.47).
Test solution. Dilute the preparation to be examined with water R to obtain a concentration of
1 mg/mL. Desalt 0.25 mL of the solution by passage through a micro-concentrator cartridge
provided with a membrane with a molecular mass cut-off of not more than 10 000 Da. Add
0.2 mL of water R to the sample and desalt again. Repeat the desalting procedure once more.
Dilute the sample with water R, determine its protein concentration as described under Tests
and adjust to a concentration of approximately 1 mg/mL with water R.
Reference solution. Dissolve the contents of a vial of erythropoietin for physicochemical
tests CRS in 0.10 mL of water R. Proceed with desalting as described for the test solution.
Capillary :
– material : uncoated fused silica ;
– size : effective length = about 100 cm, Ø = 50 µm.

CZE buffer concentrate (0.1 M sodium chloride, 0.1 M tricine, 0.1 M sodium acetate). Dissolve
0.584 g of sodium chloride R, 1.792 g of tricine R and 0.820 g of anhydrous sodium acetate R
in water R and dilute to 100.0 mL with the same solvent.
1 M putrescine solution. Dissolve 0.882 g of putrescine R in 10 mL of water R. Distribute in
0.5 mL aliquots.
CZE buffer (0.01 M tricine, 0.01 M sodium chloride, 0.01 M sodium acetate, 7 M urea, 2.5 mM
putrescine). Dissolve 21.0 g of urea R in 25 mL of water R by warming in a water-bath at
30 °C. Add 5.0 mL of CZE buffer concentrate and 125 µL of 1 M putrescine solution. Dilute to
50.0 mL with water R. Using dilute acetic acid R, adjust to pH 5.55 at 30 °C and filter through a
membrane filter (nominal pore size 0.45 µm).
Set the autosampler to store the samples at 4 °C during analysis.
Preconditioning of the capillary : rinse the capillary for 60 min with 0.1 M sodium hydroxide
filtered through a membrane filter (nominal pore size 0.45 µm) and for 60 min with CZE buffer.
Apply voltage for 12 h (20 kV).
Between-run rinsing : rinse the capillary for 10 min with water R, for 5 min with 0.1 M sodium
hydroxide filtered through a membrane filter (nominal pore size 0.45 µm) and for 10 min with
CZE buffer.
Injection : under pressure or vacuum.
Migration : apply a field strength of 143 V/cm (15.4 kV for capillaries of 107 cm total length) for
80 min, using CZE buffer as the electrolyte in both buffer reservoirs.
System suitability : in the electropherogram obtained with the reference solution, a pattern of
well separated peaks corresponding to the peaks in the electropherogram of erythropoietin
supplied with erythropoietin for physicochemical tests CRS is seen, and the largest peak is
at least 50 times greater than the baseline noise. If necessary, adjust the sample load to
give peaks of sufficient height. Identify the peaks due to isoforms 1 to 8. Isoform 1 may not
be visible. The peak due to isoform 8 is detected and the resolution between the peaks due
to isoforms 5 and 6 is not less than 1. Repeat the separation at least 3 times. The baseline
is stable, showing little drift, and the distribution of peaks is qualitatively and quantitatively
similar to the distribution of peaks in the electropherogram of erythropoietin supplied with
erythropoietin for physicochemical tests CRS. The relative standard deviation of the migration
time of the peak due to isoform 2 is less than 2 per cent.
Limits : identify the peaks due to isoforms 1 to 8 in the electropherogram obtained with the
test solution by comparison with the electropherogram obtained with the reference solution.
Calculate the percentage content of each isoform from the corresponding peak area. The
percentages are within the following ranges :
Isoform Content (per cent)
1 0 - 15
2 0 - 15
3 1 - 20
4 10 - 35
5 15 - 40
6 10 - 35
7 5 - 25
8 0 - 15
C. Polyacrylamide gel electrophoresis and immunoblotting.

(a) Polyacrylamide gel electrophoresis (2.2.31)
Gel dimensions : 0.75 mm thick, about 16 cm square.
Resolving gel : 12 per cent acrylamide.
Sample buffer : concentrated SDS-PAGE sample buffer R.

Test solution (a). Dilute the preparation to be examined in water R to obtain a concentration of
1.0 mg/mL. To 1 volume of this solution add 1 volume of sample buffer.
Test solution (b). Dilute the preparation to be examined in water R to obtain a concentration of
0.1 mg/mL. To 1 volume of this solution add 1 volume of sample buffer.
Reference solution (a). Dissolve the contents of a vial of erythropoietin for physicochemical
tests CRS in 0.10 mL of water R. To 1 volume of this solution add 1 volume of sample buffer.
Reference solution (b). Dissolve the contents of a vial of erythropoietin for physicochemical
tests CRS in water R and dilute with the same solvent to obtain a concentration of 0.1 mg/mL.
To 1 volume of this solution add 1 volume of sample buffer.
Reference solution (c). A solution of molecular mass markers suitable for calibrating
SDS-polyacrylamide gels in the range of 10-70 kDa.
Reference solution (d). A solution of pre-stained molecular mass markers suitable for
calibrating SDS-polyacrylamide gels in the range of 10-70 kDa and suitable for the
electrotransfer to an appropriate membrane.
Sample treatment : boil for 2 min.
Application : 20 µL, in the following order : reference solution (c), reference solution (a), test
solution (a), empty well, reference solution (b), test solution (b), reference solution (d).
At the end of the separation, remove the gel-cassette from the apparatus and cut the gel into 2
parts : the first part containing reference solution (c), reference solution (a) and test solution (a) ;
the second part containing reference solution (b), test solution (b) and reference solution (d).
Detection : by Coomassie staining on the first part of the gel.
System suitability : reference solution (c) :
– the validation criteria are met.
Results : the electropherogram obtained with test solution (a) shows a single diffuse band
corresponding in position and intensity to the single band seen in the electropherogram
obtained with reference solution (a).
(b) Immunoblotting
Transfer the second part of the gel onto a membrane suitable for the immobilisation of proteins,
using commercially available electrotransfer equipment and following the manufacturer’s
instructions. After electrotransfer, incubate the membrane in a neutral isotonic buffer
containing a suitable blocking agent (for example, 50 g/L of dried milk or 10 per cent V/V foetal
calf serum), for 1-2 h, followed by incubation for 1-14 h in the same blocking solution with
a suitable dilution of either a polyclonal or monoclonal anti-erythropoietin antibody. Detect
erythropoietin-bound antibody using a suitable enzyme- or radiolabelled antibody (for example,
an alkaline phosphatase-conjugated second antibody). The precise details of blocking agents,
concentrations and incubation times should be optimised using the principles set out in
Immunochemical methods (2.7.1).
System suitability : in the electropherogram obtained with reference solution (d), the molecular
mass markers are resolved on the membrane into discrete bands, with a linear relationship
between distance migrated and logarithm10 of the molecular mass.
Results : the electropherogram obtained with test solution (b) shows a single broad band
corresponding in position and intensity to the single band seen in the electropherogram
obtained with reference solution (b).
D. Peptide mapping (2.2.55). Liquid chromatography (2.2.29).
Test solution. Dilute the preparation to be examined in tris acetate buffer solution pH 8.5 R to
a concentration of 1.0 mg/mL. Equilibrate the solution in tris acetate buffer solution pH 8.5 R
using a suitable procedure (dialysis against tris acetate buffer solution pH 8.5 R, or membrane
filtration using the procedure described under Identification B, but reconstituting the desalted
sample with tris acetate buffer solution pH 8.5 R, are suitable). Transfer the dialysed solution to
a polypropylene centrifuge tube. Freshly prepare a solution of trypsin for peptide mapping R at
a concentration of 1 mg/mL in water R, and add 2 µL to 0.10 mL of the dialysed solution. Cap
the tube and place in a water-bath at 37 °C for 18 h. Remove the sample from the water-bath
and stop the reaction immediately by freezing.

Reference solution. Dissolve the contents of a vial of erythropoietin for physicochemical

tests CRS in 0.10 mL of water R. Prepare as for the test solution, ensuring that all procedures
are carried out simultaneously, and under identical conditions.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
– stationary phase : butylsilyl silica gel for chromatography R (5-10 µm).
Mobile phase :
– mobile phase A : 0.06 per cent V/V solution of trifluoroacetic acid R ;
– mobile phase B : to 100 mL of water R add 0.6 mL of trifluoroacetic acid R and dilute to
1000 mL with acetonitrile for chromatography R ;
Time Flow rate Mobile phase A Mobile phase B
(min) (mL/min) (per cent V/V) (per cent V/V)
0 - 10 0.75 100 0
10 - 125 0.75 100 → 39 0 → 61
125 - 135 1.25 39 → 17 61 → 83
135 - 145 1.25 17 → 0 83 → 100
145 - 150 1.25 100 0

Equilibration : at initial conditions for at least 15 min. Carry out a blank run using the
above-mentioned gradient.
Injection : 50 µL.
System suitability : the chromatograms obtained with the test solution and the reference
solution are qualitatively similar to the chromatogram of erythropoietin digest supplied with
erythropoietin for physicochemical tests CRS.
Results : the profile of the chromatogram obtained with the test solution corresponds to that of
the chromatogram obtained with the reference solution.
E. N-terminal sequence analysis.
The first 15 amino acids are : Ala - Pro - Pro - Arg - Leu - Ile - (no recovered peak) - Asp - Ser
- Arg - Val - Leu - Glu - Arg - Tyr.
Perform the Edman degradation using an automated solid-phase sequencer, operated in
accordance with the manufacturer’s instructions.
Desalt the equivalent of 50 µg of erythropoietin. For example, dilute a volume of the preparation
to be examined equivalent to 50 µg of the active substance in 1 mL of a 0.1 per cent V/V
solution of trifluoroacetic acid R. Pre-wash a C18 reverse-phase sample preparation cartridge
according to the instructions supplied and equilibrate the cartridge in a 0.1 per cent V/V solution
of trifluoroacetic acid R. Apply the sample to the cartridge, and wash successively with a
0.1 per cent V/V solution of trifluoroacetic acid R containing 0 per cent, 10 per cent and 50 per
cent V/V of acetonitrile R according to the manufacturer’s instructions. Lyophilise the 50 per
cent V/V acetonitrile R eluate.
Redissolve the desalted sample in 50 µL of a 0.1 per cent V/V solution of trifluoroacetic acid R
and couple to a sequencing cartridge using the protocol provided by the manufacturer. Run 15
sequencing cycles, using the reaction conditions for proline when running the 2nd and 3rd cycles.
Identify the phenylthiohydantoin (PTH)-amino acids released at each sequencing cycle by
reverse-phase liquid chromatography. The procedure may be carried out using the column and
reagents recommended by the manufacturer of the sequencing equipment for the separation
of PTH-amino-acids.
The separation procedure is calibrated using :
– the mixture of PTH-amino acids provided by the manufacturer of the sequencer, with the
gradient conditions adjusted as indicated to achieve optimum resolution of all amino acids ;
– a sample obtained from a blank sequencing cycle obtained as recommended by the
equipment manufacturer.

TESTS
Protein (2.5.33, Method I) : 80 per cent to 120 per cent of the stated concentration.
Test solution. Dilute the preparation to be examined in a 4 g/L solution of ammonium hydrogen
carbonate R.
Record the absorbance spectrum between 250 nm and 400 nm. Measure the value at the
absorbance maximum (276-280 nm), after correction for any light scattering, measured up to
400 nm. Calculate the concentration of erythropoietin taking the specific absorbance to be 7.43.
Dimers and related substances of higher molecular mass with molecular masses greater
than that of erythropoietin. Size-exclusion chromatography (2.2.30) : use the normalisation
procedure.
Test solution. Dilute the preparation to be examined in the mobile phase to obtain a concentration
of 0.2 mg/mL.
Resolution solution. Dissolve the contents of a vial of erythropoietin for SEC system suitability CRS
in the mobile phase to obtain a concentration of 0.2 mg/mL.
Reference solution. To Dilute 0.02 mL of the test resolution solution add 0.98 to 1.0 mL of with the
mobile phase.
Column :
– size : l = 0.6 0.3 m, Ø = 7.5 mm ;
– stationary phase : hydrophilic silica gel for chromatography R (5 µm), of a grade suitable for
fractionation of globular proteins in the relative molecular mass range of 20 000 to 200 000(17).
Mobile phase : dissolve 1.15 g of anhydrous disodium hydrogen phosphate R, 0.2 g of potassium
dihydrogen phosphate R and 23.4 g of sodium chloride R in 1 L of water R (1.5 mM potassium
dihydrogen phosphate, 8.1 mM disodium hydrogen phosphate, 0.4 M sodium chloride, pH 7.4) ;
adjust to pH 7.4 if necessary water R, adjust to pH 7.4 if necessary and dilute to 1 L with water R.
Injection : 100 µL.
Run time : minimum 1 h 1.5 times the retention time of erythropoietin monomer.
Relative retention with reference to erythropoietin monomer : erythropoietin dimer = about 0.9.
– the area of the principal peak in the chromatogram obtained with the reference solution is
1.5 per cent to 2.5 per cent of the area of the principal peak in the chromatogram obtained with
the test resolution solution ;
– resolution : minimum 1.5 between the peaks due to erythropoietin dimer and erythropoietin
monomer in the chromatogram obtained with the resolution solution.
Limit :
– total sum of any the peaks eluted before the principal peak : not more than the area of the
principal peak in the chromatogram obtained with the reference solution (2 per cent) maximum
2.0 per cent ; disregard any peak with a retention time greater than that of the principal peak.
(17) Tosoh TSKgel 3000SWXL is suitable.

Pharmacopoeia.
1. oligomer 2. dimer 3. monomer
Figure 1316.-1. – Chromatogram for the test for dimers and related substances with molecular
masses greater than that of erythropoietin : resolution solution
Sialic acids : minimum 10 mol of sialic acids (calculated as N-acetylneuraminic acid) per mole of
erythropoietin.
Test solution (a). Dilute the preparation to be examined in the mobile phase used in the test for
dimers and related substances of higher with molecular masses greater than that of erythropoietin
to obtain a concentration of 0.3 mg/mL.
Test solution (b). To 0.5 mL of test solution (a) add 0.5 mL of the mobile phase used in the test for
dimers and related substances of higher with molecular masses greater than that of erythropoietin.
Reference solution (a). Dissolve a suitable amount of N-acetylneuraminic acid R in water R to
obtain a concentration of 0.1 mg/mL.
Reference solution (b). To 0.8 mL of reference solution (a) add 0.2 mL of water R.
Reference solution (c). To 0.6 mL of reference solution (a) add 0.4 mL of water R.
Reference solution (d). To 0.4 mL of reference solution (a) add 0.6 mL of water R.
Reference solution (e). To 0.2 mL of reference solution (a) add 0.8 mL of water R.
Reference solution (f). Use water R.
Carry out the test in triplicate. Transfer 100 µL of each of the test and reference solutions to
10 mL glass test tubes. To each tube add 1.0 mL of resorcinol reagent R. Stopper the tubes and
incubate at 100 °C for 30 min. Cool on ice. To each tube, add 2.0 mL of a mixture of 12 volumes of
butanol R and 48 volumes of butyl acetate R. Mix vigorously, and allow the 2 phases to separate.
Ensuring that the upper phase is completely clear, remove the upper phase, taking care to exclude
completely any of the lower phase. Measure the absorbance (2.2.25) of all samples at 580 nm.
Using the calibration curve generated by the reference solutions, determine the content of sialic
acids in test solutions (a) and (b) and calculate the mean. Calculate the number of moles of sialic
acids per mole of erythropoietin assuming that the relative molecular mass of erythropoietin is 30
600 and that the relative molecular mass of N-acetylneuraminic acid is 309.
– the individual replicates agree to within ± 10 per cent of each other ;

– the value obtained from reference solution (a) is between 1.5 and 3.3 times that obtained
with test solution (a).
Bacterial endotoxins (2.6.14) : less than 20 IU in the volume that contains 100 000 IU of
erythropoietin.
ASSAY
The activity of the preparation is compared with that of erythropoietin BRP and expressed in
International Units (IU).
The estimated potency is not less than 80 per cent and not more than 125 per cent of the stated
potency. The confidence limits of the estimated potency (P = 0.95) are not less than 64 per cent
and not more than 156 per cent of the stated potency.
Carry out the determination of potency by Method A or B.
A. In polycythaemic mice
The activity of the preparation is estimated by examining, under given conditions, its effect in
stimulating the incorporation of 59Fe into circulating red blood cells of mice made polycythaemic
by exposure to reduced atmospheric pressure.
The following schedule, using treatment in a hypobaric chamber, has been found to be suitable.
Induce polycythaemia in female mice of the same strain, weighing 16-18 g. Place the mice in a
hypoxic chamber and reduce the pressure to 0.6 atmospheres. After 3 days at 0.6 atmospheres,
further reduce the pressure to 0.4-0.5 atmospheres and maintain the animals at this pressure for
a further 11 days (the partial vacuum is interrupted daily for a maximum of 1 h at about 11:00 a.m.,
in order to clean the cages and feed the animals). At the end of the specified period, return the
mice to normal atmospheric conditions. Randomly distribute the mice into cages, each containing
6 animals, and mark them.
Test solution (a). Dilute the substance to be examined in phosphate-albumin buffered saline
pH 7.2 R1 to obtain a concentration of 0.2 IU/mL.
Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline
pH 7.2 R1.
Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline
pH 7.2 R1.
Reference solution (a). Dissolve erythropoietin BRP in phosphate-albumin buffered saline
pH 7.2 R1 to obtain a concentration of 0.2 IU/mL.
Reference solution (b). Mix equal volumes of reference solution (a) and phosphate-albumin
buffered saline pH 7.2 R1.
Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin
Radiolabelled ferric [59Fe] chloride solution, concentrated. Use a commercially available solution
of [59Fe]ferric chloride (approximate specific activity : 100-1000 MBq/mg of Fe).
Radiolabelled [59Fe]ferric chloride solution. Dilute the concentrated radiolabelled [59Fe]ferric
chloride solution in Sodium citrate buffer solution pH 7.8 (0.034 M sodium citrate, 0.101 M sodium
chloride) R to obtain a solution with an activity of 3.7 × 104 Bq/mL.
The concentrations of the test solutions and reference solutions may need to be modified, based
on the response range of the animals used.
3 days after returning the animals to atmospheric pressure, inject each animal subcutaneously
with 0.2 mL of one of the solutions. The 6 animals in each cage must each receive one of the
6 different treatments (3 test solutions and 3 reference solutions), and the order of injection must
be separately randomised for each cage. A minimum of 8 cages is recommended. 2 days after
injection of the test or reference solution, inject each animal intraperitoneally with 0.2 mL of
radiolabelled [59Fe]ferric chloride solution. The order of the injections must be the same as that of
the erythropoietin injections, and the time interval between administration of the erythropoietin
and the radiolabelled ferric chloride solution must be the same for each animal. After a further

48 h, anaesthetise each animal by injection of a suitable anaesthetic, record body weights and
withdraw blood samples (0.65 mL) into haematocrit capillaries from the bifurcation of the aorta.
After determining the packed cell volume for each sample, measure the radioactivity.
Calculate the response (percentage of iron-59 in total circulating blood) for each mouse using
the expression :
As = radioactivity in the sample ;

At = total radioactivity injected ;
7.5 = total blood volume as per cent body weight ;
M = body weight, in grams ;
Vs = sample volume.
Calculate the potency by the usual statistical methods for a parallel line assay. Eliminate from the
calculation any animal where the packed cell volume is less than 54 per cent, or where the body
weight is more than 24 g.
B. In normocythaemic mice
The assay is based on the measurement of stimulation of reticulocyte production in
normocythaemic mice.
The assay may be carried out using the following procedure :
Test solution (a). Dilute the preparation to be examined in phosphate-albumin buffered saline
pH 7.2 R1 to obtain a concentration of 80 IU/mL.
Test solution (b). Mix equal volumes of test solution (a) and phosphate-albumin buffered saline
pH 7.2 R1.
Test solution (c). Mix equal volumes of test solution (b) and phosphate-albumin buffered saline
pH 7.2 R1.
Reference solution (a). Dissolve erythropoietin BRP in phosphate-albumin buffered saline
pH 7.2 R1 to obtain a concentration of 80 IU/mL.
Reference solution (b). Mix equal volumes of reference solution (a) and phosphate-albumin
Reference solution (c). Mix equal volumes of reference solution (b) and phosphate-albumin
The exact concentrations of the test solutions and reference solutions may need to be modified,
based on the response range of the animals used.
At the beginning of the assay procedure, randomly distribute mice of a suitable age and
strain (8-week old B6D2F1 mice are suitable) into 6 cages. A minimum of 8 mice per cage is
recommended. Inject each animal subcutaneously with 0.5 mL of the appropriate treatment
(one solution per cage) and put the animal in a new cage. Combine the mice in such a way that
each cage housing the treated mice contains one mouse out of the 6 different treatments (3 test
solutions and 3 reference solutions, 6 mice per cage). 4 days after the injections, collect blood
samples from the animals and determine the number of reticulocytes using a suitable procedure.
The following method may be employed :
The volume of blood, dilution procedure and fluorescent reagent may need to be modified to
ensure maximum development and stability of fluorescence.
Colorant solution, concentrated. Use a solution of thiazole orange suitable for the determination
of reticulocytes. Prepare at a concentration twice that necessary for the analysis.

Proceed with the following dilution steps. Dilute whole blood 500-fold in the buffer used to prepare
the colorant solution. Dilute this solution 2-fold in the concentrated colorant solution. After staining
for 3-10 min, determine the reticulocyte count microfluorometrically in a flow cytometer. The
percentage of reticulocytes is determined using a biparametric histogram : number of cells/red
fluorescence (620 nm).
Calculate the potency by the usual statistical methods for a parallel line assay.
STORAGE
In an airtight container at a temperature below − 20 °C. Avoid repeated freezing and thawing.
LABELLING
The label states :
– the erythropoietin content in milligrams per millilitre ;
– the activity in International Units per millilitre ;
– the name and the concentration of any other excipients.

Reference: PA/PH/Exp. 13B/T (16) 62 ANP

XXXX:1323
FENUGREEK
Trigonellae foenugraeci semen

DEFINITION
Dried, ripe seeds of Trigonella foenum-graecum L.
CHARACTERS
Strong characteristic aromatic odour.
IDENTIFICATION
A. The seed is hard, flattened, brown or reddish-brown and more or less rhomboidal with rounded
edges. It is 3-5 mm long, 2-3 mm wide and 1.5-2 mm thick. The widest surfaces are marked by
a groove that divides the seed into 2 unequal parts. The smaller part contains the radicle ; the
larger part contains the cotyledons.
B. Microscopic examination (2.8.23). The powder is yellowish-brown. Examine under a
microscope using chloral hydrate solution R. The powder shows the following diagnostic
characters (Figure 1323.-1) : fragments of the testa (transverse section [B]) with lageniform
epidermal cells [Bb] covered by a thick cuticle [Ba], a hypodermis consisting of large cells,
narrower at the upper end and constricted in the middle, with bar-like thickenings of the radial
walls [Bc] and parenchyma with flattened cells [Bd] ; fragments of the epidermis in surface view,
yellowish-brown, consisting of small polygonal cells, either with thick, channelled walls and a
narrow lumen (view from above [G]) or with smooth walls and a larger lumen (view from below
[C]) ; fragments of the hypodermis in surface view, with cells having either a circular outline
and thickened walls, closely beaded (view from above [H]), or having a polyhedral outline
whose bar-like thickenings extend from the lower to the upper walls (view from below [A]) ;
parenchyma of the testa consisting of loosely arranged cells leaving numerous spaces [E] ;
fragments of endosperm with rounded [F] or elongated [D] cells, associated with mucilage [Fa]
depending on the orientation, and sometimes with small spiral or annular vessels [J].
Figure 1323.-1. – Illustration for identification test B of powdered herbal drug of fenugreek
PA/PH/Exp. 13B/T (16) 62 ANP

Test solution. Place 1.0 g of the powdered herbal drug (710) (2.9.12) in a 25 mL conical flask
and add 5.0 mL of methanol R. Heat in a water-bath at 65 °C for 5 min. Cool and filter.
Reference solution. Dissolve 3.0 mg of trigonelline hydrochloride R in 1.0 mL of methanol R.
Plate : TLC silica gel F254 plate R.
Mobile phase : water R, methanol R (30:70 V/V).
Application : 20 µL of the test solution and 10 µL of the reference solution, as bands.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the chromatogram obtained with the test solution shows in its lower half a quenching
zone similar in position and fluorescence to the zone in the chromatogram obtained with the
reference solution.
Detection B : spray with potassium iodobismuthate solution R2.
Results B : the chromatogram obtained with the test solution shows an intense orange-red
zone similar in position and colour to the zone in the chromatogram obtained with the reference
solution. It also shows in its upper half, a broad light brownish-yellow zone (triglycerides).
TESTS
Swelling index (2.8.4) : minimum 6, determined on the powdered herbal drug (710) (2.9.12).


Definition : upper and lower content limits modified in accordance with the Technical guide when
alkalimetric titrations with potentiometric end-point determinations are prescribed.
Appearance of solution : test replaced by a more accurate absorbance test as there is evidence
that the colour of solution is due to very low concentrations of a number of highly coloured
degradation compounds.
Related substances, Impurity F : both replaced by a single, more sensitive LC method ; limits
updated in view of recent batch data. An isocratic step before the start of the gradient programme
has not been introduced as this negatively affects the selectivity of the method. The correction
factors for the coeluting impurities C and I are 0.4 and 2.0 respectively, so to calculate their sum, a
correction factor of 2.0 is applied even though this may lead to an overestimation.
Impurities : impurity D deleted as not detected in any batch, and impurity F now listed as
impurities H and I.
XXXX:1693
FLUPENTIXOL DIHYDROCHLORIDE
Flupentixoli dihydrochloridum
C23H27Cl2F3N2OS Mr 507.4
[2413-38-9]
DEFINITION
2-[4-[3-[(EZ)-2-(Trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]piperazin-1-yl]ethan-1-ol
dihydrochloride.
Content :
– flupentixol dihydrochloride : 98.0 99.0 per cent to 101.5 101.0 per cent (dried substance) ;
– (Z)-isomer : 42.0 per cent to 52.0 per cent.
CHARACTERS
Appearance : white or almost white powder.
Solubility : very soluble in water, soluble in ethanol (96 per cent), practically insoluble in methylene
chloride.
IDENTIFICATION
First identification : A, D.
Second identification : B, C, D.
Comparison : flupentixol dihydrochloride CRS.
B. Thin-layer chromatography (2.2.27).
Test solution. Dissolve 20 mg of the substance to be examined in methanol R and dilute to
Reference solution. Dissolve 20 mg of flupentixol dihydrochloride CRS in methanol R and

Mobile phase : water R, diethylamine R, methyl ethyl ketone R (1:4:95 V/V/V).
Development : twice over a path of 15 cm 3/4 of the plate.
Drying : in air.
Detection A : examine in ultraviolet light at 254 nm.
Results A : the principal spot in the chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram obtained with the reference solution.
Doubling of the spot may be observed in both chromatograms.
Detection B : spray with alcoholic solution of sulfuric acid R ; heat at 110 °C for 5 min and allow
to cool ; examine in ultraviolet light at 365 nm.
Results B : the principal spot in the chromatogram obtained with the test solution is similar in
Doubling of the spot may be observed in both chromatograms.
C. Mix about 5 mg with 45 mg of heavy magnesium oxide R and ignite in a crucible until an
almost white residue is obtained (usually less than 5 min). Allow to cool, add 1 mL of water R,
0.05 mL of phenolphthalein solution R1 and about 1 mL of dilute hydrochloric acid R to render
the solution colourless. Filter and add to the filtrate a freshly prepared mixture of 0.1 mL of
alizarin S solution R and 0.1 mL of zirconyl nitrate solution R. Mix, allow to stand for 5 min and
compare the colour of the solution with that of a blank prepared in the same manner. The test
solution is yellow. The blank is red.
D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than
reference solution GY6 (2.2.2, Method II).
Dissolve 2.0 g of the substance to be examined in water R and dilute to 20 mL with the same
solvent.
pH (2.2.3) : 2.0 to 3.0.
Dissolve 0.5 g in carbon dioxide-free water R and dilute to 50 mL with the same solvent.
Absorbance (2.2.25) : maximum 0.125 at 410 nm.
Dissolve 0.5 g in 5.0 mL of water R.

Related substances. Liquid chromatography (2.2.29). Carry out the test protected from light and
prepare the solutions immediately before use.

Pharmacopoeia.
1. impurity A 2. impurity H 3. impurities C + I 4. flupentixol (Z-isomer) 5. flupentixol

(E-isomer)
6. impurity B 7. impurity B 8. impurity G 9. impurity E (isomer 1) 10. impurity E
(isomer 1) (isomer 2) (isomer 2)
Figure 1693.-1. – Chromatogram for the test for related substances of flupentixol dihydrochloride :
test solution spiked with impurities A, B, C, E, G, H and I
Buffer solution. Dissolve 6.3 g of ammonium formate R in 900 mL of water for chromatography R,
adjust to pH 8.2 with concentrated ammonia R and dilute to 1000 mL with water for
chromatography R.
Test solution. Dissolve 58.0 mg of the substance to be examined in mobile phase A and dilute to
50.0 mL with mobile phase A.
Reference solution (a). Dilute 1.0 mL of the test solution to 100.0 mL with mobile phase A. Dilute
1.0 mL of this solution to 10.0 mL with mobile phase A.
Reference solution (b). Dissolve 2 mg of flupentixol for system suitability CRS (containing
impurities A, C, H and I) in mobile phase A and dilute to 10 mL with mobile phase A.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
Mobile phase :
(18) Gemini-NX C18 is suitable.

– mobile phase A : buffer solution, acetonitrile for chromatography R, water for chromatography R
(58:420:522 V/V/V) ;
– mobile phase B : buffer solution, acetonitrile for chromatography R (100:900 V/V) ;
0 - 40 90 → 5 10 → 95

Injection : 20 µL.
Identification of impurities : use the chromatogram supplied with flupentixol for system
suitability CRS and the chromatogram obtained with reference solution (b) to identify the peaks
due to impurities A, C + I, and H.
Relative retention with reference to flupentixol (Z)-isomer (retention time = about 12 min) :
impurity A = about 0.83 ; impurity H = about 0.89 ; impurities C and I = about 0.97 ; flupentixol
(E)-isomer = about 1.04.
– resolution : minimum 2.5 between the peaks due to impurities A and H ;
– peak-to-valley ratio : minimum 10.0, where Hp = height above the baseline of the peak due to
impurities C and I and Hv = height above the baseline of the lowest point of the curve separating
this peak from the peak due to flupentixol (Z)-isomer.
– correction factor : multiply the peak area of impurities C and I by 2.0 ;
– for each impurity, use the concentration of flupentixol in reference solution (a).
Limits :
– impurity H : maximum 0.5 per cent ;
– sum of impurities C and I : maximum 0.3 per cent ;
Related substances. Thin-layer chromatography (2.2.27). Carry out the test protected from light
and prepare the solutions immediately before use.
Test solution (a). Dissolve 0.40 g of the substance to be examined in ethanol (96 per cent) R and
Test solution (b). Dilute 2.0 mL of test solution (a) to 20.0 mL with ethanol (96 per cent) R.
Reference solution (a). Dilute 1.0 mL of test solution (b) to 50.0 mL with ethanol (96 per cent) R.
Reference solution (b). Dilute 2.0 mL of reference solution (a) to 20.0 mL with ethanol (96 per
cent) R.
Reference solution (c). Dissolve 10 mg of flupentixol impurity D CRS in ethanol (96 per cent) R,
add 0.5 mL of test solution (a) and dilute to 20.0 mL with ethanol (96 per cent) R.
Mobile phase : diethylamine R, toluene R, ethyl acetate R (10:20:70 V/V/V).
Development : in an unsaturated tank over a path of 10 cm.
Drying : in air.
Detection : spray with alcoholic solution of sulfuric acid R, heat at 110 °C for 5 min and allow to cool ;
examine in ultraviolet light at 365 nm. Doubling of the spot due to flupentixol may be observed.
System suitability : the chromatogram obtained with reference solution (c) shows 2 clearly
separated spots.

Limits :
– in the chromatogram obtained with test solution (a) : any spots, apart from the principal spot,
are not more intense than the spot, or spots in the chromatogram obtained with reference
solution (a) (0.2 per cent) ;
– in the chromatogram obtained with test solution (b) : any spots, apart from the principal spot,
are not more intense than the spot or spots in the chromatogram obtained with reference
solution (b) (0.2 per cent).
Impurity F. Liquid chromatography (2.2.29). Carry out the test protected from light and prepare
the solutions immediately before use.
Test solution. Dissolve 20.0 mg of the substance to be examined in the mobile phase and dilute
to 20.0 mL with the mobile phase.
Reference solution. Dissolve 10.0 mg of flupentixol dihydrochloride CRS and 10.0 mg of
flupentixol impurity F CRS in the mobile phase and dilute to 100.0 mL with the mobile phase.
Column :
– size : l = 0.125 m, Ø = 4.6 mm ;
– stationary phase : octylsilyl silica gel for chromatography R (3 µm)(20).
Mobile phase : mix 10 volumes of acetonitrile R, 55 volumes of methanol R and 35 volumes of
a solution containing 8.72 g/L of potassium dihydrogen phosphate R, 0.37 g/L of anhydrous
disodium hydrogen phosphate R and 0.77 g/L of dodecyltrimethylammonium bromide R.
Injection : 20 µL.
– resolution : minimum 2.0 between the 2nd of the peaks due to impurity F and the 1st of the peaks
due to flupentixol ; peak splitting may not always occur.
Limit :
– impurity F : not more than the area of the corresponding peak or peaks in the chromatogram
obtained with the reference solution (0.5 per cent).
Loss on drying (2.2.32) : maximum 2.0 per cent, determined on 1.000 g by drying in an oven
at 105 °C.
Sulfated ash (2.4.14) : maximum 0.1 per cent, determined on 1.0 g in a platinum crucible.
ASSAY
Flupentixol dihydrochloride. Dissolve 0.200 g in 30 mL of ethanol (96 per cent) R. Carry out a
potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read the volume added between
the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 50.74 mg of C23H27Cl2F3N2OS.
(Z)-Isomer. Liquid chromatography (2.2.29).
Reference solution. Dissolve 20.0 mg of flupentixol dihydrochloride CRS in the mobile phase
and dilute to 50.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.0 mm ;
– stationary phase : silica gel for chromatography R (5 µm).
Mobile phase : water R, concentrated ammonia R, 2-propanol R, heptane R (2:4:150:850 V/V/V/V).
(20) Spherisorb C8 is suitable.

Injection : 20 µL.
– resolution : minimum 3.0 between the peaks due to (Z)-isomer (1st peak) and to (E)-isomer
(2nd peak).
Results :
– calculate the percentage content of (Z)-isomer taking into account the assigned content of
(Z)-isomer in flupentixol dihydrochloride CRS ;
– calculate the ratio of the area of the peak due to the (E)-isomer to the area of the peak due to
the (Z)-isomer : this ratio is 0.9 to 1.4.
STORAGE
IMPURITIES
Specified impurities : C, H, I.
for pharmaceutical use) : A, B, E, G.
A. (9RS)-9-[3-(dimethylamino)propyl]-2-(trifluoromethyl)-9H-thioxanthen-9-ol,
B. N,N-dimethyl-3-[(EZ)-2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propan-1-amine,
C. 1-[3-[(EZ)-2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]piperazine,
D. 2-[2-[4-[3-[(EZ)-2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]piperazin-1-
yl]ethoxy]ethan-1-ol,

E. 2-[4-[3-[(EZ)-2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]piperazin-1-yl]ethyl acetate,
F. mixture of 2-[4-[(E)-3-[(9RS)-2-(trifluoromethyl)-9H-thioxanthen-9-yl]prop-2-enyl]piperazin-1-
yl]ethanol and 2-[4-[(Z)-3-[(9RS)-2-(trifluoromethyl)-9H-thioxanthen-9-yl]prop-2-enyl]piperazin-
1-yl]ethanol,
G. 2-(trifluoromethyl)-9H-thioxanthen-9-one,
H, I. for each impurity, either 2-[4-[(E)-3-[(9RS)-2-(trifluoromethyl)-9H-thioxanthen-9-yl]prop-

2-enyl]piperazin-1-yl]ethan-1-ol or 2-[4-[(Z)-3-[(9RS)-2-(trifluoromethyl)-9H-thioxanthen-9-
yl]prop-2-enyl]piperazin-1-yl]ethan-1-ol.

Reference: PA/PH/Exp. EXT/T (16) 2 ANP

The definition of the Drug Extract Ratio (DER) was amended to improve the differentiation
between the DERgenuine and the DERtotal.
XXXX:0765
HERBAL DRUG EXTRACTS
Plantarum medicinalium extracta

DEFINITION
Herbal drug extracts are liquid (liquid extraction preparations), semi-solid (soft extracts and
oleoresins) or solid (dry extracts) preparations obtained from Herbal drugs (1433) using suitable
solvents.
An extract is essentially defined by the quality of the herbal drug, by its production process
(extraction solvent(s), method of processing, etc.) and by its specifications.
European Pharmacopoeia monographs for extracts cover the genuine (native) extract and, where
present, excipients.
Different types of extract may be distinguished.
Standardised extracts are adjusted to a defined content of one or more constituents with
known therapeutic activity. This is achieved by adjustment of the extract with inert excipients or
by blending batches of the extract.
Quantified extracts are adjusted to one or more active markers, the content of which is controlled
within a limited, specified range. Adjustments are made by blending batches of the extract.
Other extracts are not adjusted to a particular content of constituents. For control purposes, one
or more constituents are used as analytical markers. The minimum content for these analytical
markers is given in an individual monograph.
PRODUCTION
Herbal drugs, solvents and other materials used for the preparation of extracts are of suitable
quality and where applicable comply with the requirements of any relevant monograph in the
European Pharmacopoeia. Where justified, herbal drugs used for the production of extracts may
exceed the limits for heavy metals specified in the monograph Herbal drugs (1433) provided that
the resulting extract satisfies the requirements for heavy metals (see Tests).
Different batches of the herbal drug which are compliant with the relevant monograph, or in the
absence of an individual monograph with other suitable specifications, may be combined prior to
extraction, for example for the purpose of achieving the quantity of herbal drug required for the
production process or, in the case of standardised and quantified extracts, to achieve a certain
range of content for one or more constituents in the herbal drug to be extracted. The herbal drug
may also undergo a preliminary treatment, for example, grinding, inactivation of enzymes or
defatting. In addition, unwanted constituents (e.g. toxic constituents) or unwanted matter (e.g.
insoluble matter) may be removed at a suitable stage in the production process.
Where solvents are recovered from the production process, such recovered or recycled solvents
may be used, provided that the recovery procedures are controlled and monitored to ensure that
solvents meet appropriate standards before re-use or admixture with other approved materials.
Water used for the production of extracts complies with the requirements of the monograph Water
for preparation of extracts (2249).
Where applicable, miscella (extraction liquors) are concentrated to the intended consistency using
suitable methods, usually under reduced pressure and at a temperature at which deterioration
of the constituents is reduced to a minimum. Essential oils that have been separated during
processing may be restored to the extracts at an appropriate stage in the production process.
Suitable excipients may be added at various stages of the production process for technological
reasons (for example, as part of the drying process or to improve the homogeneity or consistency

of an extract). For standardised extracts, suitable inert excipients may also be added to adjust
one or more constituents to a defined content. For quantified extracts and ‘other’ extracts,
the addition of inert excipients to adjust the content of assayed constituents is not permitted.
Excipients are included for technological reasons only, and the manufacturer must declare the
content of such excipients as a fixed percentage. In some applications, an excipient may be
added in a narrow percentage range (e.g. silicon dioxide between 0.1-0.5 per cent, to improve
flowability of the extract). The proposed range must be justified by the manufacturer. Suitable
stabilisers, antioxidants and antimicrobial preservatives may be added to extracts where justified
and authorised.
Extraction with a given solvent leads to a typical content of selected constituents in the extracted
dry matter ; during production of standardised and quantified extracts, purification procedures may
be applied that increase the content of these selected constituents with respect to the expected
values ; such extracts are referred to as ‘refined’.
IDENTIFICATION
Extracts are identified using suitable methods.
TESTS
Where applicable, as a result of analysis of the herbal drug used for production and in view of
the production process, tests for microbiological quality (5.1.4 or 5.1.8), heavy metals (2.4.27),
aflatoxins (2.8.18), ochratoxin A (2.8.22) and pesticide residues (2.8.13) in the extracts may be
necessary. Where a test for heavy metals is carried out, the same limits for heavy metals as those
given in the monograph Herbal drugs (1433) are applicable to extracts unless otherwise stated in
an individual extract monograph or unless otherwise justified and authorised.
ASSAY
Extracts are assayed by a suitable method, unless otherwise justified.
Standardised extracts. The Definition section of an individual monograph on a standardised
extract states the content of the assayed constituents as either a defined single content or within a
defined range of content.
Defined single content. For example, in the monograph Ipecacuanha liquid extract, standardised
(1875), the content of assayed constituents is stated as 1.80 per cent to 2.20 per cent. In this
case, the declaration is based on a defined single content of 2.0 per cent with a tolerance of
± 10 per cent. The acceptable tolerance is usually within the range ± 5 per cent to ± 10 per cent
taking into account the nature of the extract and the method of assay.
Defined range of content. For example, in the monograph Frangula bark dry extract, standardised
(1214), the content of assayed constituents is stated as 15.0 per cent to 30.0 per cent. In this
case, it is intended that an extract will consistently be produced to a defined single content
selected from within the defined range taking into account an acceptable tolerance. Where there
is an individual monograph in the pharmacopoeia for a standardised extract with a defined range
of content, the acceptable tolerance will be stated in the individual monograph (for example, for
Frangula bark dry extract, standardised (1214), the acceptable tolerance is stated as ± 10 per
cent relative to the declared content).
Quantified extracts. The content of assayed constituents must be within the values given in the
Definition section of an individual monograph.
Other extracts. The content of assayed constituents must not be lower than the minimum value
given in the Definition section of an individual monograph. Where justified and authorised,
this does not preclude the selection of alternative constituents as a basis for assay using a
corresponding validated analytical method, which may be more appropriate to the physical and/or
chemical properties of the medicinal product into which the extract is to be incorporated. Where
alternative constituents are selected for assay, a suitable minimum value for such constituents
must be established.
LABELLING
The label states :

– the herbal drug used ;

– where applicable, that fresh herbal drug has been used ;
– the form of the extract (for example, liquid, tincture, soft, oleoresin or dry) ;
– where applicable, that the extract is standardised or quantified ;
– for standardised extracts, the defined content of constituents with known therapeutic activity ;
– for quantified extracts, the specified range of content of active markers ;
– where applicable, that the extract is ‘refined’ ;
– the first solvent or solvents used for extraction (for example, ethanol 60 per cent V/V) ;
– the name and amount of any excipients present in the extract (for example, diluents, stabilisers,
antimicrobial preservatives, antioxidants) ;
– for quantified extracts and ‘other’ extracts, the ratio of the quantity of herbal drug to the quantity
of genuine (native) extract (DERgenuine) expressed on a mass/mass basis for soft extracts,
oleoresins and dry extracts, and on either a mass/mass or a mass/volume basis for liquid
extraction preparations ;
– where applicable, the percentage of dry residue ;
– the storage conditions.
Liquid extraction preparations - Praeparationes fluidae ab extractione

Liquid extraction preparations are liquid preparations consisting of a diverse range of products
which are described by their extraction solvents, methods of production and drug solvent ratios or
drug extract ratios. Included in this range are products obtained using ethanol, water, glycerol,
propylene glycol and fatty oils as extraction solvents. Liquid (fluid) extracts and tinctures belong to
this category and are described below.
LIQUID (FLUID) EXTRACTS – EXTRACTA FLUIDA

DEFINITION
Quantified liquid (fluid) extracts and ‘other’ liquid (fluid) extracts are liquid extraction preparations
of which, in general, 1 part by mass or volume is equivalent to 1 part by mass of the dried herbal
drug.
Standardised liquid (fluid) extracts are only defined by their content of constituents with known
therapeutic activity.
PRODUCTION
Liquid extracts are prepared using ethanol of a suitable concentration and/or water together with,
where necessary, other substances (e.g. glycerol or ammonia solution) to extract the herbal drug,
or by dissolving a soft or dry extract of the herbal drug (which has been produced using the same
extraction solvent as would be used to prepare the liquid extract by direct extraction) in either
ethanol of the required concentration or water.
Where the liquid extract contains ethanol, it is tested for 2-propanol (2.9.11), with a maximum
of 0.05 per cent V/V, unless assurance of compliance with this limit is provided by a detailed
knowledge of the ethanol supply chain and the extract manufacturing process.
Except for standardised liquid extracts, liquid extracts produced from soft or dry extracts do not
contain any excipients other than those that would be present in the liquid extract prepared
by direct extraction. However, exceptions may be justified in certain cases such as when the
soft extract used to produce the liquid extract contains stabilisers, antioxidants or antimicrobial
preservatives that have been added to ensure its stability.
Liquid extracts are adjusted, if necessary, so that they satisfy the requirements for content of
solvent. Liquid extracts may be filtered, if necessary.
A slight sediment may form on standing.
TESTS
Relative density (2.2.5). Where applicable, the liquid extract complies with the limits prescribed.

Ethanol (2.9.10). For ethanolic liquid extracts, carry out the determination of ethanol content.
The ethanol content complies with the limits prescribed.
Methanol (2.9.11) : maximum 0.05 per cent V/V for ethanolic liquid extracts, unless otherwise
prescribed or justified and authorised.
Dry residue (2.8.16). Where applicable, the liquid extract complies with the limits prescribed.
STORAGE
LABELLING
The label states in addition to the requirements listed above, the ethanol content in per cent V/V,
where applicable.
TINCTURES – TINCTURAE
DEFINITION
Quantified tinctures and ‘other’ tinctures are liquid extraction preparations that are obtained using
either 1 part by mass of herbal drug and 10 parts by mass or volume of extraction solvent, or
1 part by mass of herbal drug and 5 parts by mass or volume of extraction solvent. Alternatively,
they may be obtained using either 1 part by mass of herbal drug and sufficient extraction solvent
to produce 10 parts by mass or volume of tincture or 1 part by mass of herbal drug and sufficient
extraction solvent to produce 5 parts by mass or volume of tincture. Other ratios of herbal drug
to extraction solvent may be used.
Standardised tinctures are only defined by their content of constituents with known therapeutic
activity.
PRODUCTION
Tinctures are usually prepared by either maceration or percolation, using ethanol of a suitable
concentration to extract the herbal drug, or by dissolving a soft or dry extract of the herbal drug
(which has been produced using the same extraction solvent as would be used to prepare the
tincture by direct extraction) in ethanol of the required concentration.
The tincture is tested for 2-propanol (2.9.11), with a maximum of 0.05 per cent V/V, unless
assurance of compliance with this limit is provided by a detailed knowledge of the ethanol supply
chain and the tincture manufacturing process.
Except for standardised tinctures, tinctures produced from soft or dry extracts do not contain any
excipients other than those that would be present in the tincture prepared by direct extraction.
However, exceptions may be justified in certain cases such as when the soft extract used to
produce the tincture contains stabilisers, antioxidants or antimicrobial preservatives that have
been added to ensure its stability.
Tinctures are adjusted, if necessary so that they satisfy the requirements for content of solvent.
Tinctures may be filtered if necessary.
Tinctures are usually clear. A slight sediment may form on standing.
TESTS
Relative density (2.2.5). Where applicable, the tincture complies with the limits prescribed.
Ethanol (2.9.10). The ethanol content complies with the limits prescribed.
Methanol (2.9.11) : maximum 0.05 per cent V/V, unless otherwise prescribed or justified and
authorised.
Dry residue (2.8.16). Where applicable, the tincture complies with the limits prescribed.
STORAGE
LABELLING
The label states, in addition to the requirements listed above,
the ethanol content in per cent V/V.

Soft extracts – extracta spissa

DEFINITION
Soft extracts are semi-solid preparations obtained by evaporation or partial evaporation of the
solvent used for production.
TESTS
Dry residue (2.8.16). The soft extract complies with the limits prescribed.
Solvents. Residual solvents are controlled as described in chapter 5.4, unless otherwise
STORAGE
Oleoresins – oleoresina
DEFINITION
Oleoresins are semi-solid extracts composed of a resin in solution in an essential and/or fatty oil
and are obtained by evaporation of the solvent(s) used for their production.
This monograph applies to oleoresins produced by extraction and not to natural oleoresins.
TESTS
Water (2.2.13). The oleoresin complies with the limits prescribed.
STORAGE
Dry extracts – extracta sicca

DEFINITION
Dry extracts are solid preparations obtained by evaporation of the solvent used for their production.
Dry extracts usually have a loss on drying of not greater than 5 per cent m/m. Where justified and
authorised, a loss on drying with a different limit or a test for water may be prescribed.
TESTS
Loss on drying (2.8.17). Where applicable, the dry extract complies with the limits prescribed.
Water (2.5.12). Where a test for loss on drying is not applicable, the dry extract complies with
the limits prescribed.
STORAGE
Glossary - glossa
Constituents with known therapeutic activity. Chemically defined substances or groups of
substances which are generally accepted to contribute substantially to the therapeutic activity of a
herbal drug, a herbal drug preparation or a herbal medicinal product.
Drug extract ratio (DER). The ratio between the quantity of herbal drug used in the manufacture
of an extract and the quantity of extract obtained. The number (given as the actual range) written
before the colon is the relative quantity of the herbal drug ; the number written after the colon is the
relative quantity of the extract obtained. Two DERs can be differentiated :

– Genuine (native) drug extract ratio (DERgenuine). See under drug extract ratio (DER) for
the explanation of how the ratio for a DER is designated. A DERgenuine The ratio between the
quantity of herbal drug used in the manufacture of an extract and the quantity of genuine
(native) extract obtained. The number (given as the actual range) written before the colon
is the relative quantity of the herbal drug ; the number written after the colon is the relative
quantity of the genuine extract obtained.
– Total drug extract ratio (DERtotal). The ratio between the quantity of herbal drug used in the
manufacture of an extract and the quantity of whole extract (including excipients) obtained.
For example, DERgenuine 2.5-4.5 :1 means that between 2.5 and 4.5 parts of herbal drug are
required to produce 1 part of genuine (native) extract. Where processing aids are added to the
genuine (native) extract to produce, for example, a dry extract, the DER DERtotal and the DERgenuine
will have different values ; where a dry extract is produced without the need for any processing
aids, the DER DERtotal and the DERgenuine will be identical. Oleoresins are usually produced without
the need to include processing aids, therefore the DER DERtotal and the DERgenuine are usually
identical. For soft extracts and liquid extraction preparations, where the genuine (native) extract
does not exist without excipients and/or processing aids (e.g. usually 20-30 per cent of water in
soft extracts, ethanolic extraction solvent in tinctures), the DER DERtotal and the DERgenuine are
identical.
Drug solvent ratio (DSR). The ratio between the quantity of herbal drug, expressed in mass,
used in the manufacture of an extract and the quantity of the first extraction solvent, expressed in
mass or volume.
Extraction solvents. Solvents which are used for the extraction process.
Genuine (native) herbal drug extract. Refers to the extract without excipients, even if for
technological reasons the genuine extract is not available. However, for soft extracts and liquid
extraction preparations the genuine extract may contain variable amounts of (extraction) solvent.
Markers. Chemically defined constituents or groups of constituents of a herbal drug, a herbal drug
preparation or a herbal medicinal product which are of interest for control purposes independent
of whether they have any therapeutic activity. Markers serve to calculate the quantity of herbal
drug(s) or herbal drug preparation(s) in the herbal medicinal product if the marker has been
quantitatively determined in the herbal drug or herbal drug preparation.
There are 2 categories of markers :
– active markers are constituents or groups of constituents which are generally accepted to
contribute to the therapeutic activity ;
– analytical markers are constituents or groups of constituents that serve solely for analytical
purposes, irrespective of any pharmacological or therapeutic activity which they may be
reported to possess.
Miscella (extraction liquor). Liquid obtained from the extraction process.
Production of tinctures by maceration. A process whereby, unless otherwise prescribed,
the herbal drug to be extracted is reduced to pieces of suitable size, mixed thoroughly with the
prescribed extraction solvent and allowed to stand in a closed container for an appropriate
time, with agitation where required. The residue is separated from the extraction solvent and, if
necessary, pressed out. If the residue is pressed, the 2 liquids are combined.
Production of tinctures by percolation. A process whereby, unless otherwise prescribed,
the herbal drug to be extracted is reduced to pieces of suitable size and mixed thoroughly with
a portion of the prescribed extraction solvent and allowed to stand for an appropriate time. The
mixture is transferred to a percolator and more extraction solvent is added until the herbal drug is
covered with a layer of extraction solvent. The percolate is allowed to flow slowly from the base
of the percolator while extraction solvent is slowly added to the top of the percolator, ensuring
that the herbal drug to be extracted is constantly covered with extraction solvent, until all the
extraction solvent has been added. Percolation continues until the percolate is recovered. If the
residue is pressed, the 2 liquids are combined.

Reference: PA/PH/Exp. TCM/T (16) 144 ANP
XXXX:2566
ISATIS ROOT
Isatidis radix
Banlangen
板蓝根
DEFINITION
Dried root of Isatis tinctoria L. (I. indigotica Fortune) collected in autumn.
Content : minimum 1.0 per cent of arginine (C6H14N4O2 ; Mr 174.2) (dried drug).
IDENTIFICATION
A. The root is cylindrical, slightly tortuous, 10-20 cm long, 0.5-1 cm in diameter, externally
greyish-yellow or brownish-yellow, wrinkled longitudinally and lenticellate transversally, with
rootlets or rootlet scars. Root stock slightly expanded, exhibiting dark green or dark brown
petiole bases arranged in whorls, and dense tubercles. The fracture is yellowish-white, brown
or dark brown in bark and yellow or brown in wood.
B. Microscopic examination (2.8.23). The powder is whitish-yellow or yellow. Examine under

a microscope using chloral hydrate solution R. The powder shows the following diagnostic
characters : fragments of cork consisting of 5-8 thin-walled layers ; fragments of xylem with
reticulate structure ; thin-walled, rounded parenchyma cells. Examine under a microscope
using a 50 per cent V/V solution of glycerol R. The powder shows abundant, single or
compound (2, 3 or 4) starch grains. The starch grains, 1.5-3.4 µm in diameter, with spot, cleft
or V-shaped hilum.
B. Microscopic examination (2.8.23). The powder is whitish-yellow or yellow. Examine under

a microscope using chloral hydrate solution R. The powder shows the following diagnostic
characters (Figure 2566.-1) : fragments of cork consisting of 5-8 layers of thin-walled cells
(surface view [A], transverse section [B]) ; fragments of xylem [D] consisting of reticulate or
pitted vessels [Da] included in thin-walled parenchyma cells [Db]; thin-walled parenchyma
cells (longitudinal section [E], transverse section [F]). Examine under a microscope using a
50 per cent V/V solution of glycerol R. The powder shows abundant, single or compound (2,
3 or 4) starch grains, 1.5-3.4 µm in diameter, free [C] or included in parenchyma [G], with a
punctiform, slit-shaped or V-shaped hilum.

Figure 2566.-1. – Illustration for identification test B of powdered herbal drug of isatis root
Test solution. To 0.5 g of the powdered herbal drug (355) (2.9.12) add 5 mL of ethanol (70 per
cent V/V) R and sonicate for 10 min. Centrifuge and use the supernatant.
Reference solution. Dissolve 4 mg of arginine R and 4 mg of cysteine hydrochloride R in 1 mL
of ethanol (70 per cent V/V) R.
Plate : TLC silica gel F254 plate R (5-40 µm) [or TLC silica gel F254 plate R (2-10 µm)].
Mobile phase : anhydrous formic acid R, water R, acetonitrile R (2:8:30 V/V/V).
Application : 4 µL as bands of 10 mm [or 8 mm].
Development : over a path of 8.5 cm [or 6 cm].
Drying : in air.
Detection : expose to concentrated ammonia R vapour for 5 min, treat with ninhydrin
solution R4, then heat at 120 °C for 3 min.
reference solution and the test solution. Furthermore, other faint coloured zones may be
present in the chromatogram obtained with the test solution.
Top of the plate
_______ _______
_______ _______
A prominent brown zone

Cysteine : a brown zone
A brown zone
Arginine : a brown zone A brown zone (arginine)

A faint brown zone
TESTS
drug (355) (2.9.12) by drying in an oven at 105 °C for 2 h.

Ash insoluble in hydrochloric acid (2.8.1) : maximum 1.0 per cent.
ASSAY
Pharmacopoeia.
1. cysteine 2. arginine
Figure 2566.-2. – Chromatogram for the assay of isatis root : reference solution (b)
Test solution. To 0.100 g of the powdered herbal drug (355) (2.9.12) add 20 mL of ethanol (70 per
cent V/V) R, sonicate for 20 min, filter, and evaporate the filtrate to dryness. Dissolve the residue
in ethanol (70 per cent V/V) R and dilute to 10.0 mL with the same solvent.
Reference solution (a). Dissolve 25.0 mg of arginine CRS in ethanol (70 per cent V/V) R and dilute
to 50.0 mL with the same solvent.
Reference solution (b). Dissolve 3.0 mg of cysteine hydrochloride R in 6.0 mL of reference
solution (a) and dilute to 10.0 mL with ethanol (70 per cent V/V) R.
Reference solutions (c), (d), (e), (f), (g), (h). Dilute reference solution (a) to obtain 6 reference
solutions of arginine, the concentrations of which span the expected value in the test solution.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
Mobile phase : trifluoroacetic acid R, water R (0.2:99.8 V/V).
Detection : evaporative light-scattering detector ; the following settings have been found to be
suitable ; if the detector has different setting parameters, adjust the detector settings so as to
comply with the system suitability criterion for the signal-to-noise ratio :
– carrier gas : nitrogen R ;
– pressure : 330 kPa ;
– evaporator temperature : 80 °C.
Injection : 10 µL.
Run time : 25 min.
– resolution : minimum 1.5 between the peaks due to cysteine and arginine in the chromatogram
obtained with reference solution (b) ;
– signal-to-noise ratio : minimum 50 for the peak due to arginine in the chromatogram obtained
with reference solution (a).
(21) Inertsil ODS-3 is suitable.

Establish a calibration curve with the logarithm of the concentration (in milligrams per 10 mL) of
reference solutions (c), (d), (e), (f), (g) and (h) (corrected by the assigned percentage content of
arginine CRS) as the abscissa and the logarithm of the corresponding peak areas as the ordinate.
Calculate the percentage content of arginine using the following expression :
A = logarithm of the concentration of arginine in the test solution, determined from

the calibration curve ;
m = mass of the herbal drug to be examined used to prepare the test solution, in
grams.


Characters : statement on polymorphism added.
Identification : recrystallisation step added in identification A due to the presence of different
crystalline forms and description of the sample preparation deleted in accordance with current
policy ; ether replaced by 1,1-dimethylethyl methyl ether in identification B ; former identification
C by melting point deleted.
Related substances : identification of impurities and relative retention introduced, and limits
updated in line with current batch data ; explicit acceptance criterion for unspecified impurities
introduced and the disregard limit modified to be in line with the general monograph Substances
for Pharmaceutical Use (2034).
Impurity A : old colorimetric test not sensitive enough at 100 ppm, so replaced by a new GC
method with a tighter limit.
XXXX:1242
MEPIVACAINE HYDROCHLORIDE
Mepivacaini hydrochloridum
C15H23ClN2O Mr 282.8
[1722-62-9]
DEFINITION
(RS)-N-(2,6-Dimethylphenyl)-1-methylpiperidine-2-carboxamide hydrochloride.
CHARACTERS
Appearance : white or almost white, crystalline powder.
Solubility : freely soluble in water and in ethanol (96 per cent), very slightly soluble in methylene
chloride.
mp : about 260 °C, with decomposition.
It shows polymorphism (5.9).
IDENTIFICATION
First identification : A, B, D, C.
Second identification : B, C, D.
Preparation : discs.
Comparison : mepivacaine hydrochloride CRS.
If the spectra obtained in the solid state show differences, dissolve the substance to be
examined and the reference substance separately in methanol R, evaporate to dryness and
dry in an oven at 80 °C for 45 min. Record new spectra using the residues.
Test solution. Dissolve 20 mg of the substance to be examined in ethanol (96 per cent) R and
Reference solution (a). Dissolve 20 mg of mepivacaine hydrochloride CRS in ethanol (96 per
cent) R and dilute to 5 mL with the same solvent.

Reference solution (b). Dissolve 20 mg of mepivacaine hydrochloride CRS and 20 mg of

lidocaine hydrochloride CRS in ethanol (96 per cent) R and dilute to 5 mL with the same solvent.
Mobile phase : concentrated ammonia R, methanol R, ether R1,1-dimethylethyl methyl ether R
(1:5:100 V/V/V).
Development : over a path of 12 cm2/3 of the plate.
Drying : in air.
Detection : examine in ultraviolet light at 254 nm.
– the chromatogram shows 2 clearly separated principal spots.
Results : the principal spot in the chromatogram obtained with the test solution is similar in
position and size to the principal spot in the chromatogram obtained with reference solution (a).
C. To 5 mL of solution S (see Tests) add 1 mL of dilute sodium hydroxide solution R and shake
with 2 quantities, each of 10 mL, of ether R. Dry the combined upper layers over anhydrous
sodium sulfate R. Filter and evaporate the ether on a water-bath. Dry the residue at 100-105 °C
for 2 h. The melting point (2.2.14) is 151 °C to 155 °C.
C. D. It gives reaction (a) of chlorides (2.3.1).
TESTS
Solution S. Dissolve 1.5 g in carbon dioxide-free water R and dilute to 30 mL with the same
solvent.
Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than
reference solution B7 (2.2.2, Method II).
pH (2.2.3) : 4.0 to 5.0.
Dilute 2 mL of solution S to 5 mL with carbon dioxide-free water R.
Optical rotation (2.2.7) : − 0.10° to + 0.10°, determined on solution S.
Reference solution (a). Dissolve 20.0 mg of the substance to be examined and 30.0 mg of
mepivacaine impurity B CRS in the mobile phase and dilute to 100.0 mL with the mobile phase.
Reference solution (b). Dilute 1.0 mL of the test solution to 100.0 mL with the mobile phase.
Dilute 1.0 mL of this solution to 10.0 mL with the mobile phase.
Column :
– size : l = 0.125 m, Ø = 4.6 mm ;
– stationary phase : base-deactivated octadecylsilyl silica gel for chromatography R (5 µm)(22).
Mobile phase : mix 35 volumes of acetonitrile R1 and 65 volumes of a 2.25 g/L solution of
phosphoric acid R adjusted to pH 7.6 with strong sodium hydroxide solution R.
Flow rate : 1 mL/min.
Injection : 20 µL.
Run time : 3 times the retention time of mepivacaine.
the peak due to impurity B.
Relative retention with reference to mepivacaine (retention time = about 7 min) : impurity B = about
0.5.
(22) Genesis C18 is suitable.

– resolution : minimum 2.5 between the peaks due to impurity B and mepivacaine.
Limits :
– impurities B, C, D, E : for each impurity, not more than twice the area of the principal peak in
the chromatogram obtained with reference solution (b) (0.2 per cent), and not more than one
of the peaks has an area greater than the area of the principal peak in the chromatogram
obtained with reference solution (b) (0.1 per cent) ;
chromatogram obtained with reference solution (b) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.5 per cent) ;
– disregard limit : 0.2 0.5 times the area of the principal peak in the chromatogram obtained with
reference solution (b) (0.02 0.05 per cent).
Pharmacopoeia.
1. impurity B 2. mepivacaine
Figure 1242.-1. – Chromatogram for the test for related substances of mepivacaine hydrochloride :
reference solution (a)

Pharmacopoeia.
1. impurity A
Figure 1242.-2. – Chromatogram for the test for impurity A of mepivacaine hydrochloride : test
solution spiked with impurity A
Impurity A. Head-space gas chromatography (2.2.28).
Test solution. Introduce 60.0 mg of the substance to be examined into a 20 mL vial. Add 2.0 mL
of a 103.0 g/L solution of hydrochloric acid R and 1.0 mL of a 126.0 g/L solution of sodium
hydroxide R and close the vial immediately.
Reference solution. Dissolve 6.0 mg of bupivacaine impurity F CRS (impurity A) in a 103.0 g/L
solution of hydrochloric acid R and dilute to 100.0 mL with the same solvent. Dilute 1.0 mL of this
solution to 100.0 mL with the same solvent. Introduce 2.0 mL of this solution into a 20 mL vial, add
1.0 mL of a 126.0 g/L solution of sodium hydroxide R and close the vial immediately.
Column :
– material : fused silica ;
– size : l = 30 m, Ø = 0.53 mm ;
Carrier gas : helium for chromatography R.
Split ratio : 1:1.
– pressurisation time : 2 min.
Temperature :
(23) Agilent DB624 is suitable.

Time Temperature
(min) (°C)
Column 0 - 10 130 → 230
10 - 15 230
Injection port 225
Detector 250

Retention time : impurity A = about 6 min.
– repeatability : maximum relative standard deviation of 15.0 per cent determined on 6 injections.
Calculation of content :
– for impurity A, use the concentration of impurity A in the reference solution.
Limit :
– impurity A : maximum 20 ppm.
Impurity A : maximum 100 ppm.
Dissolve 0.50 g in methanol R and dilute to 10 mL with the same solvent. To 2 mL of this solution
add 1 mL of a freshly prepared 10 g/L solution of dimethylaminobenzaldehyde R in methanol R
and 2 mL of glacial acetic acid R and allow to stand for 10 min. Any yellow colour in the solution is
not more intense than that in a standard prepared at the same time and in the same manner using
2 mL of a 5 mg/L solution of 2,6-dimethylaniline R in methanol R.
at 105 °C.
ASSAY
Dissolve 0.250 g in a mixture of 5.0 mL of 0.01 M hydrochloric acid and 50 mL of ethanol (96 per
cent) R. Carry out a potentiometric titration (2.2.20), using 0.1 M sodium hydroxide. Read
the volume added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 28.28 mg of C15H23ClN2O.
IMPURITIES
Specified impurities : A, B, C, D, E.
for pharmaceutical use) : B, C, D, E.
A. 2,6-dimethylaniline,
B. (RS)-N-(2,6-dimethylphenyl)piperidine-2-carboxamide,

C. N-(2,6-dimethylphenyl)pyridine-2-carboxamide,
D. (RS)-N-(2,6-dimethylphenyl)-1-methyl-1,2,5,6-tetrahydropyridine-2-carboxamide,
E. (RS)-N-(4-chloro-2,6-dimethylphenyl)-1-methylpiperidine-2-carboxamide.

Reference: PA/PH/Exp. 13B/T (16) 60 ANP

Water : it is proposed to replace the test for loss on drying by the determination of water content
as the latter has been found more suitable.
XXXX:2071
MILK THISTLE DRY EXTRACT, REFINED AND STANDARDISED
Silybi mariani extractum siccum raffinatum et normatum

DEFINITION
Dry extract, refined and standardised, produced from Milk thistle fruit (1860).
Content : 90 per cent to 110 per cent of the nominal content of silymarin, expressed as silibinin
(C25H22O10 ; Mr 482.4), stated on the label. The nominal content of silymarin is within the range
30 per cent m/m to 65 per cent m/m (dried anhydrous extract).
The content of silymarin corresponds to :
– sum of the contents of silicristin and silidianin (both C25H22O10 ; Mr 482.4) : 20 per cent to 45 per
cent, calculated with reference to total silymarin ;
– sum of the contents of silibinin A and silibinin B (both C25H22O10 ; Mr 482.4) : 40 per cent to
65 per cent, calculated with reference to total silymarin ;
– sum of the contents of isosilibinin A and isosilibinin B (both C25H22O10 ; Mr 482.4) : 10 per cent to
20 per cent, calculated with reference to total silymarin.
PRODUCTION
The extract is produced from the herbal drug by an appropriate procedure, using one or more of
the following solvents :
– ethyl acetate ;
– acetone or mixture of acetone and water ;
– ethanol or mixture of ethanol and water ;
– methanol or mixture of methanol and water.
CHARACTERS
Appearance : yellowish-brown, amorphous powder.
IDENTIFICATION
Test solution. Dissolve 0.250 g of the extract to be examined in 5 mL of methanol R.
Reference solution. Dissolve 2 mg of silibinin R and 5 mg of taxifolin R in 10 mL of methanol R.
Plate : TLC silica gel plate R (5-40 µm) [or TLC silica gel plate R (2-10 µm)].
Mobile phase : anhydrous formic acid R, acetone R, methylene chloride R (8.5:16.5:75 V/V/V).
Application : 10 µL [or 8 µL] of the test solution and 10 µL [or 2 µL] of the reference solution,
as bands.
Drying : at 100-105 °C.
Detection : treat the still-warm plate with a 10 g/L solution of diphenylboric acid aminoethyl ester R
in methanol R and subsequently treat with a 50 g/L solution of macrogol 400 R in methanol R.
Allow to dry for 30 min and examine in ultraviolet light at 365 nm.
reference solution and the test solution. Furthermore, other yellowish-green fluorescent zones
may be present between the zones due to silibinin and taxifolin in the chromatogram obtained
with the test solution.

Top of the plate

Silibinin : a yellowish-green fluorescent zone A yellowish-green fluorescent zone (silibinin)
____ ____
Taxifolin : an orange fluorescent zone An orange fluorescent zone (taxifolin)

A yellowish-green fluorescent zone (silicristin)
____ ____
A fluorescent zone (line of application)

TESTS
Loss on drying (2.8.17) : maximum 5.0 per cent.
Water (2.5.12) : maximum 4.0 per cent, determined on 0.500 g(24).
ASSAY
Pharmacopoeia.
1. silicristin 3. silibinin A 5. isosilibinin A

2. silidianin 4. silibinin B (+ minor shoulder : dihydrosilibinin B) 6. isosilibinin B
Figure 2071.-1. – Chromatogram for the assay of refined and standardised milk thistle dry extract
Test solution. Dissolve 60.0 mg of the extract to be examined in methanol R and dilute to 100.0 mL
Reference solution. Dissolve a quantity of milk thistle dry extract HRS corresponding to 10.0 mg
of silibinin in methanol R and dilute to 100.0 mL with the same solvent.
Column :
– size : l = 0.125 m, Ø = 4 mm ;
(24) Hydranal Composite 5 (Riedel de Haën) is suitable.

(25) Hypersil ODS C18 is suitable.

Mobile phase :
– mobile phase A : phosphoric acid R, methanol R, water for chromatography R (0.5:35:65 V/V/V) ;
– mobile phase B : phosphoric acid R, methanol R, water for chromatography R (0.5:50:50 V/V/V) ;
Time Mobile phase A Mobile phase B
0 - 28 100 → 0 0 → 100
28 - 35 0 100
35 - 36 0 → 100 100 → 0
36 - 51 100 0

Injection : 10 µL.
Identification of peaks : use the chromatogram supplied with milk thistle dry extract HRS and
the chromatogram obtained with the reference solution to identify the peaks due to silicristin,
silidianin, silibinin A, silibinin B, isosilibinin A and isosilibinin B. In the chromatogram obtained with
the test solution the peak due to silidianin may vary in size or be absent.
Retention time : silibinin B = about 30 min ; if necessary, adjust the time periods of the gradient.
– resolution : minimum 1.8 between the peaks due to silibinin A and silibinin B ;
– the chromatogram is similar to the chromatogram supplied with milk thistle dry extract HRS.
Calculate the percentage content of total silymarin, expressed as silibinin, using the following
expression :
Calculate the percentage content of the sum of silicristin and silidianin, with reference to total
silymarin, using the following expression :
Calculate the percentage content of the sum of silibinin A and silibinin B, with reference to total
silymarin, using the following expression :
Calculate the percentage content of the sum of isosilibinin A and isosilibinin B, with reference to
total silymarin, using the following expression :
A1 = area of the peak due to silicristin in the chromatogram obtained with the test
solution ;
A2 = area of the peak due to silidianin in the chromatogram obtained with the test
solution ;
A3 = area of the peak due to silibinin A in the chromatogram obtained with the test
solution ;
A4 = area of the peak due to silibinin B in the chromatogram obtained with the test
solution ;

A5 = area of the peak due to isosilibinin A in the chromatogram obtained with the test
solution ;
A6 = area of the peak due to isosilibinin B in the chromatogram obtained with the test
solution ;
A7 = area of the peak due to silibinin A in the chromatogram obtained with the
reference solution ;
A8 = area of the peak due to silibinin B in the chromatogram obtained with the
reference solution ;
m1 = mass of milk thistle dry extract HRS used to prepare the reference solution, in
grams ;
m2 = mass of the extract to be examined used to prepare the test solution, in grams ;
p = combined percentage content of silibinin A and silibinin B in milk thistle dry
extract HRS.


Definition : lower and upper content limits modified to reflect change in assay method.
Solubility : grade of ethanol specified.
Identification B : chloroform replaced by methylene chloride.
Related substances : TLC method replaced with LC method capable of controlling 6 unspecified
impurities.
Isomer test : deleted as impurity E is now controlled by the related substances test.
Assay : titrimetric method using pyridine and end-point determination by colour indicator replaced
with LC method used in related substances test.
Impurities : section introduced.
XXXX:0200
PENTOBARBITAL
Pentobarbitalum
C11H18N2O3 Mr 226. 3
[76-74-4]
DEFINITION
5-Ethyl-5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione.
Content : 99.0 98.0 per cent to 101.0 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or colourless crystals.
Solubility : very slightly soluble in water, freely soluble in anhydrous ethanol. It forms water-soluble
compounds with alkali hydroxides and carbonates and with ammonia.
IDENTIFICATION
A. Determine the melting point (2.2.14) of the substance to be examined. Mix equal parts of the
substance to be examined and pentobarbital CRS and determine the melting point of the
mixture. The difference between the melting points (which are about 133 °C) is not greater
than 2 °C.
B. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R as the coating
substance.
Test solution. Dissolve 0.1 g of the substance to be examined in alcohol R and dilute to 100 mL
Reference solution. Dissolve 0.1 g of pentobarbital CRS in alcohol R and dilute to 100 mL
Apply to the plate 10 µL of each solution. Develop over a path of 18 cm using the lower layer of
a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes
of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal spot in the
chromatogram obtained with the test solution is similar in position and size to the principal spot
in the chromatogram obtained with the reference solution.
Test solution. Dissolve 10 mg of the substance to be examined in ethanol (96 per cent) R
and dilute to 10 mL with the same solvent.

Reference solution. Dissolve 10 mg of pentobarbital CRS in ethanol (96 per cent) R and dilute
to 10 mL with the same solvent.
Mobile phase : concentrated ammonia R, ethanol (96 per cent) R, methylene chloride R
(5:15:80 V/V/V) ; use the lower layer.
Development : over 3/4 of the plate.
Detection : examine immediately in ultraviolet light at 254 nm.
C. To about 10 mg add about 10 mg of vanillin R and 2 mL of sulfuric acid R. Mix and heat on
a water-bath for 2 min. A reddish-brown colour develops. Cool and add cautiously 5 mL of
anhydrous ethanol R. The colour becomes violet and then blue.
TESTS
Appearance of solution. The solution is clear (2.2.1) and not more intensely coloured than
reference solution Y6 (2.2.2, Method II).
Dissolve 1.0 g in a mixture of 4 mL of dilute sodium hydroxide solution R and 6 mL of water R.
Acidity. Boil 1.0 g with 50 mL of water R for 2 min, allow to cool and filter. To 10 mL of the filtrate
add 0.15 mL of methyl red solution R. The solution is orange-yellow. Not more than 0.1 mL of
0.1 M sodium hydroxide is required to produce a pure yellow colour.
Related substances. Examine by thin-layer chromatography (2.2.27), using silica gel GF254 R
as the coating substance.
Reference solution. Dilute 0.5 mL of the test solution to 100 mL with alcohol R.
Apply to the plate 20 µL of each solution. Develop over a path of 15 cm using the lower layer of a
mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and 80 volumes of
chloroform R. Examine immediately in ultraviolet light at 254 nm. Spray with diphenylcarbazone
mercuric reagent R. Allow the plate to dry in air and spray with freshly prepared alcoholic
potassium hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R. Heat at 100 °C to
105 °C for 5 min and examine immediately. When examined in ultraviolet light and after spraying,
any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not
more intense than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Test solution (a). Dissolve 25.0 mg of the substance to be examined in the mobile phase and
dilute to 25.0 mL with the mobile phase.
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL with the mobile phase.
Reference solution (a). Dilute 1.0 mL of test solution (b) to 100.0 mL with the mobile phase.
Reference solution (b). Dissolve 2.5 mg of pentobarbital impurity E CRS in the mobile phase and
dilute to 25.0 mL with the mobile phase. Dilute 1.0 mL of the solution to 100.0 mL with the mobile
phase. Dissolve 5 mg of the substance to be examined in 5.0 mL of this solution.
Reference solution (c). Dissolve 25.0 mg of pentobarbital CRS in the mobile phase and dilute to
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
potassium dihydrogen phosphate R adjusted to pH 3.5 with dilute phosphoric acid R.
(26) Zorbax Eclipse XDB-C18, Inertsil ODS-3V and Waters XBridge C18 are suitable.


Injection : 10 µL of test solution (a) and reference solutions (a) and (b).
Run time : 2.5 times the retention time of pentobarbital.
Identification of impurities : use the chromatogram obtained with reference solution (b) to identify
the peak due to impurity E.
Relative retention with reference to pentobarbital (retention time = about 10 min) :
impurity E = about 0.92.
– resolution : minimum 2.0 between the peaks due to impurity E and pentobarbital.
– for each impurity, use the concentration of pentobarbital in reference solution (a).
Limits :
Pharmacopoeia.
1. impurity A 5. impurity E
2. impurity B 6. pentobarbital
3. impurity C 7. impurity F
4. impurity D
Figure 0200.-1. – Chromatogram for the test for related substances of pentobarbital: test
solution (a) spiked with impurities A to F
Isomer. Dissolve 0.3 g in 5 mL of a 50 g/L solution of anhydrous sodium carbonate R, heating
slightly if necessary. Add a solution of 0.3 g of nitrobenzyl chloride R in 10 mL of alcohol R and
heat under a reflux condenser for 30 min. Cool to 25 °C, filter and wash the precipitate with five
quantities, each of 5 mL, of water R. In a small flask, heat the precipitate with 25 mL of alcohol R
under a reflux condenser until dissolved (about 10 min). Cool to 25 °C, if necessary scratching
the wall of the flask with a glass rod to induce crystallisation, and filter. The precipitate, washed
with two quantities, each of 5 mL, of water R and dried at 100 °C to 105 °C for 30 min, melts
(2.2.14) at 136 °C to 148 °C.
at 105 °C.

ASSAY
Dissolve 0.100 g in 5 mL of pyridine R. Add 0.5 mL of thymolphthalein solution R and 10 mL of
silver nitrate solution in pyridine R. Titrate with 0.1 M ethanolic sodium hydroxide until a pure blue
colour is obtained. Carry out a blank titration.
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to 11.31 mg of C11H18N2O3.
modification.
Injection : test solution (b) and reference solution (c).
Calculate the percentage content of C11H18N2O3 taking into account the assigned content of
pentobarbital CRS.
IMPURITIES
pharmaceutical use) : A, B, C, D, E, F.
A. (5RS)-2,6-diamino-5-ethyl-5-[(2RS)-pentan-2-yl]pyrimidin-4(5H)-one,
B. (5RS)-6-amino-5-ethyl-5-[(2RS)-pentan-2-yl]pyrimidine-2,4(3H,5H)-dione,
C. 5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione,
D. 5-methyl-5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione,
E. 5-ethyl-5-(pentan-3-yl)-1,3-diazinane-2,4,6-trione,

F. 5-ethyl-5-[(2RS)-4-methylpentan-2-yl]-1,3-diazinane-2,4,6-trione.


Definition : lower and upper content limits modified to reflect change in assay method.
Identification B : chloroform replaced by methylene chloride.
Related substances : TLC method replaced with LC method capable of controlling 6 unspecified
impurities.
Isomer test : deleted as impurity E is now controlled by the related substances test.
Assay : titrimetric method using pyridine and end-point determination by colour indicator replaced
with LC method used in related substances test.
Impurities : section introduced.
XXXX:0419
PENTOBARBITAL SODIUM
Pentobarbitalum natricum
C11H17N2NaO3 Mr 248.3
[57-33-0]
DEFINITION
Sodium derivative of 5-ethyl-5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione.
Content : 99.0 98.0 per cent to 101.5 102.0 per cent (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder, hygroscopic.
Solubility : very soluble in water.
IDENTIFICATION
A. Dissolve 1 g in 10 mL of water R and add 5 mL of dilute acetic acid R. A white, crystalline
precipitate is formed. Filter, wash the precipitate with water R and dry at 100-105 °C.
Determine the melting point (2.2.14) of the precipitate. Mix equal parts of the precipitate and
pentobarbital CRS and determine the melting point of the mixture. The difference between the
melting points (which are about 131 °C) is not greater than 2 °C.
B. Examine by thin-layer chromatography (2.2.27), using a TLC silica gel GF254 plate R.
Test solution. Dissolve 25 mg of the precipitate obtained in identification test A in alcohol R
Reference solution. Dissolve 25 mg of pentobarbital CRS in alcohol R and dilute to 25 mL
Apply to the plate 10 µL of each solution. Develop over a path of 18 cm using the lower
layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and
80 volumes of chloroform R. Examine immediately in ultraviolet light at 254 nm. The principal
spot in the chromatogram obtained with the test solution is similar in position and size to the
principal spot in the chromatogram obtained with the reference solution.
Test solution. Dissolve 11 mg of the substance to be examined in ethanol (96 per cent) R
Reference solution. Dissolve 10 mg of pentobarbital CRS in ethanol (96 per cent) R and dilute
to 10 mL with the same solvent.


Mobile phase : concentrated ammonia R, ethanol (96 per cent) R, methylene chloride R
(5:15:80 V/V/V) ; use the lower layer.
Detection : examine immediately in ultraviolet light at 254 nm.
C. To about 10 mg add about 10 mg of vanillin R and 2 mL of sulfuric acid R. Mix and heat on
a water-bath for 2 min. A reddish-brown colour develops. Cool and add cautiously 5 mL of
anhydrous ethanol R. The colour becomes violet and then blue.
D. Ignite 1 g. The residue gives reaction (a) of sodium (2.3.1).
TESTS
pH (2.2.3). 9.6 to 11.0, measured immediately after preparation.
Dissolve 1.0 g in carbon dioxide-free water R and dilute to 10 mL with the same solvent.
Related substances. Examine by thin-layer chromatography (2.2.27), using a TLC silica
gel GF254 plate R.
Reference solution. Dilute 0.5 mL of the test solution to 100 mL with alcohol R.
Apply to the plate 10 µL of each solution. Develop over a path of 15 cm using the lower
layer from a mixture of 5 volumes of concentrated ammonia R, 15 volumes of alcohol R and
80 volumes of chloroform R. Examine immediately in ultraviolet light at 254 nm. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with the reference solution (0.5 per cent). Spray
with diphenylcarbazone mercuric reagent R. Allow the plate to dry in air and spray with freshly
prepared alcoholic potassium hydroxide solution R diluted 1 in 5 with aldehyde-free alcohol R.
Heat at 100 °C to 105 °C for 5 min and examine immediately in daylight. Any spot in the
chromatogram obtained with the test solution, apart from the principal spot, is not more intense
than the spot in the chromatogram obtained with the reference solution (0.5 per cent).
Reference solution (a). Dilute 1.0 mL of test solution (b) to 100.0 mL with the mobile phase.
Reference solution (b). Dissolve 2.5 mg of pentobarbital impurity E CRS in the mobile phase and
dilute to 25.0 mL with the mobile phase. Dilute 1.0 mL of the solution to 100.0 mL with the mobile
phase. Dissolve 5 mg of the substance to be examined in 5.0 mL of this solution.
Reference solution (c). Dissolve 25.0 mg of pentobarbital CRS in the mobile phase and dilute to
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
potassium dihydrogen phosphate R adjusted to pH 3.5 with dilute phosphoric acid R.
Injection : 10 µL of test solution (a) and reference solutions (a) and (b).
Run time : 2.5 times the retention time of pentobarbital.
(27) Zorbax Eclipse XDB-C18, Inertsil ODS-3V and Waters XBridge C18 are suitable.

Identification of impurities : use the chromatogram obtained with reference solution (b) to identify
the peak due to impurity E.
Relative retention with reference to pentobarbital (retention time = about 10 min) :
impurity E = about 0.92.
– resolution : minimum 2.0 between the peaks due to impurity E and pentobarbital.
– for each impurity, use the concentration of pentobarbital sodium in reference solution (a).
Limits :
Pharmacopoeia.
1. impurity A 3. impurity C 5. impurity E 7. impurity F

2. impurity B 4. impurity D 6. pentobarbital
Figure 0419.-1. – Chromatogram for the test for related substances of pentobarbital sodium :
test solution (a) spiked with impurities A to F
Free pentobarbital : maximum 3.5 per cent.
Dissolve 2.00 g in 75 mL of dimethylformamide R, heating gently if necessary. Titrate with 0.1 M
sodium methoxide until the colour changes from olive-green to blue, using 0.25 mL of a 10 g/L
solution of thymol blue R in dimethylformamide R as indicator. Carry out a blank titration.
1 mL of 0.1 M sodium methoxide is equivalent to 22.63 mg of pentobarbital.
Isomer. Dissolve 0.3 g in 5 mL of a 50 g/L solution of anhydrous sodium carbonate R. Add a
solution of 0.3 g of nitrobenzyl chloride R in 10 mL of alcohol R and heat under a reflux condenser
for 30 min. Cool to 25 °C, if necessary scratching the wall of the container with a glass rod
to induce crystallisation. Filter and wash the precipitate with five quantities, each of 5 mL, of
water R. In a small flask, heat the precipitate with 25 mL of alcohol R under a reflux condenser
until dissolved (about 10 min). Cool to 25 °C, if necessary scratching the wall of the flask with a
glass rod to induce crystallisation, and filter. The precipitate, washed with two quantities, each of
5 mL, of water R and dried at 100 °C to 105 °C for 30 min, melts (2.2.14) at 136 °C to 148 °C.
at 105 °C.

ASSAY
Dissolve 0.200 g in 15 mL of a 127.5 g/L solution of silver nitrate R in pyridine R. Titrate with 0.1 M
ethanolic sodium hydroxide until a pure blue colour is obtained, using 0.5 mL of thymolphthalein
solution R as indicator. Carry out a blank titration.
1 mL of 0.1 M ethanolic sodium hydroxide is equivalent to 24.83 mg of C11H17N2NaO3.
modification.
Injection : test solution (b) and reference solution (c).
Calculate the percentage content of C11H17N2NaO3 taking into account the assigned content of
pentobarbital CRS and a conversion factor of 1.097.
STORAGE
IMPURITIES
pharmaceutical use) : A, B, C, D, E, F.
A. (5RS)-2,6-diamino-5-ethyl-5-[(2RS)-pentan-2-yl]pyrimidin-4(5H)-one,
B. (5RS)-6-amino-5-ethyl-5-[(2RS)-pentan-2-yl]pyrimidine-2,4(3H,5H)-dione,
C. 5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione,
D. 5-methyl-5-[(2RS)-pentan-2-yl]-1,3-diazinane-2,4,6-trione,
E. 5-ethyl-5-(pentan-3-yl)-1,3-diazinane-2,4,6-trione,

F. 5-ethyl-5-[(2RS)-4-methylpentan-2-yl]-1,3-diazinane-2,4,6-trione.


Content : lower and upper limits updated in line with current policy for assaying substances
with LC.
Characters : degree of hygroscopicity updated in line with results obtained in recent experimental
tests.
Related substances : test solution (b) and reference solution (d) introduced to allow same
method to be used in related substances test and assay.
Assay : titration replaced by LC ; potentiometric assay did not prove to be sufficiently robust
and also used a toxic reagent, i.e. pyridine.
XXXX:0521
PHENYTOIN SODIUM
Phenytoinum natricum
C15H11N2NaO2 Mr 274.3
[630-93-3]
DEFINITION
Sodium 4-oxo-5,5-diphenyl-4,5-dihydro-1H-imidazol-2-olate.
Content : 98.5 per cent to 100.5 per cent 98.0 per cent to 102.0 per cent (anhydrous substance).
CHARACTERS
Appearance : white or almost white, slightly very hygroscopic, crystalline powder.
Solubility : soluble in water and in ethanol (96 per cent), practically insoluble in methylene chloride.
IDENTIFICATION
First identification : A, C.
Second identification : B, C.
Preparation : suspend 0.1 g in 20 mL of water R. Acidify with dilute hydrochloric acid R and
shake with 3 quantities, each of 30 mL, of ethyl acetate R. Wash the combined ethyl acetate
layers with water R, evaporate to dryness and dry the residue at 100-105 °C (test residue).
Repeat the operations using 0.1 g of phenytoin sodium CRS (reference residue). Examine as
discs prepared using potassium bromide R.
Comparison : phenytoin sodium CRS.
Solvent mixture : acetone R, methanol R (50:50 V/V).
Test solution. Dissolve 20 mg of the substance to be examined in the solvent mixture and dilute
to 10 mL with the solvent mixture.
Reference solution. Dissolve 20 mg of phenytoin sodium CRS in the solvent mixture and dilute
to 10 mL with the solvent mixture.
Mobile phase : concentrated ammonia R, toluene R, 2-propanol R (10:40:50 V/V/V).
Application : 10 µL as bands of 8 mm.
Drying : at 80 °C for 5 min ; allow to cool.


Results : the principal zone in the chromatogram obtained with the test solution is similar in
position and size to the principal zone in the chromatogram obtained with the reference solution.
C. Ignite 1 g and cool. Add 2 mL of water R to the residue and neutralise the solution with
hydrochloric acid R. Filter and dilute the filtrate to 4 mL with water R. 0.1 mL of the solution
gives reaction (b) of sodium (2.3.1).
TESTS
Appearance of solution. Suspend 1.0 g in 5 mL of water R and dilute to 20 mL with 0.1 M
sodium hydroxide. The solution is clear (2.2.1) and not more intensely coloured than reference
solution BY6 (2.2.2, Method II).
Pharmacopoeia.
1. impurity C 2. impurity D 3. impurity E 4. phenytoin 5. impurity F 6. impurity A 7. impurity B
Figure 0521.-1. – Chromatogram for the test for related substances : solution of phenytoin spiked
with impurities A to F
Reference solution (a). Dilute 1.0 mL of the test solution (a) to 100.0 mL with the mobile phase.
Dilute 1.0 mL of this solution to 10.0 mL with the mobile phase.
Reference solution (b). Dissolve 2 mg of 2,2-diphenylglycine R (impurity C) in 100.0 mL of the
mobile phase.
Reference solution (c). Dissolve 10 mg of phenytoin for system suitability CRS (containing
impurities D and E) in the mobile phase, add 1.0 mL of reference solution (b) and dilute to 10.0 mL
with the mobile phase.
Reference solution (d). Dissolve 50.0 mg of phenytoin sodium CRS in the mobile phase and dilute
to 50.0 mL with the mobile phase. Dilute 1.0 mL of the solution to 20.0 mL with the mobile phase.
Column :
– size : l = 0.25 m, Ø = 4.6 mm ;
(28) YMC Pack ODS AQ 18 is suitable.

Mobile phase : mix 20 volumes of methanol R2, 35 volumes of acetonitrile R1 and 45 volumes of a
5.75 g/L solution of ammonium dihydrogen phosphate R adjusted to pH 2.5 with phosphoric acid R.
Injection : 20 µL of the test solution (a) and reference solutions (a) and (c).
Run time : 4 times the retention time of phenytoin.
Identification of impurities : use the chromatogram supplied with phenytoin for system
suitability CRS and the chromatogram obtained with reference solution (c) to identify the peaks
due to impurities C, D and E.
Relative retention with reference to phenytoin (retention time = about 4 min) : impurity C = about 0.5 ;
impurity D = about 0.6 ; impurity E = about 0.8.
System suitability : reference solution (c) :
– resolution : minimum 3.5 between the peaks due to impurities D and E.
Limits :
– correction factors : for the calculation of content, multiply the peak areas of the following
impurities by the corresponding correction factor : impurity D = 1.7 ; impurity E = 1.4 ;
– impurity E : not more than 3 times the area of the principal peak in the chromatogram obtained
with reference solution (a) (0.3 per cent) ;
– impurity C : not more than twice the area of the principal peak in the chromatogram obtained
– impurity D : not more than 1.5 times the area of the principal peak in the chromatogram obtained
chromatogram obtained with reference solution (a) (0.10 per cent) ;
– total : not more than 5 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.5 per cent) ;
– disregard limit : 0.5 times the area of the principal peak in the chromatogram obtained with
reference solution (a) (0.05 per cent).
Free phenytoin. Dissolve 0.30 g in 10 mL of a mixture of equal volumes of pyridine R and water R.
Add 0.5 mL of phenolphthalein solution R and 3 mL of silver nitrate solution in pyridine R. Not more
than 1.0 mL of 0.1 M sodium hydroxide is required to change the colour of the indicator to pink.
ASSAY
Suspend 0.180 g in 2 mL of water R. Add 8.0 mL of 0.05 M sulfuric acid and heat gently for 1 min.
Add 30 mL of methanol R and cool. Carry out a potentiometric titration (2.2.20), using 0.1 M
sodium hydroxide. After reaching the 1st point of inflexion, interrupt the addition of 0.1 M sodium
hydroxide, add 5 mL of silver nitrate solution in pyridine R, mix and continue the titration. Read
the volume of 0.1 M sodium hydroxide added between the 2 points of inflexion.
1 mL of 0.1 M sodium hydroxide is equivalent to 27.43 mg of C15H11N2NaO2.
modification.
Injection : test solution (b) and reference solution (d).
Calculate the percentage content of C15H11N2NaO2 taking into account the assigned content
of phenytoin sodium CRS.

Pharmacopoeia.
1. phenytoin
Figure 0521.-2. – Chromatogram for the assay of phenytoin sodium : test solution (b)
STORAGE
IMPURITIES
Specified impurities : C, D, E.
pharmaceutical use) : A, B, F.
A. diphenylmethanone (benzophenone),
B. diphenylethanedione (benzil),
C. amino(diphenyl)acetic acid (2,2-diphenylglycine),

D. 3a,6a-diphenyltetrahydroimidazo[4,5-d]imidazole-2,5(1H,3H)-dione,
E. (carbamoylamino)(diphenyl)acetic acid,
F. 5-(4-methylphenyl)-5-phenylimidazolidine-2,4-dione.


Identifications A and B : reaction (a) is sufficient.
Assay : with the addition of methanol to the sample solution a slight turbidity develops, which
may influence the results ; a replacement method is proposed, the sample is passed through a
strongly acidic ion-exchange column and the eluent is titrated with sodium hydroxide, avoiding
the use of lead nitrate.
XXXX:1622
POTASSIUM SULFATE
Kalii sulfas
K2SO4 Mr 174.3
[7778-80-5]
DEFINITION
Content : 98.5 per cent to 101.0 per cent of K2SO4 (dried substance).
CHARACTERS
Appearance : white or almost white, crystalline powder or colourless crystals.
Solubility : soluble in water, practically insoluble in ethanol.
IDENTIFICATION
A. It gives the reactions reaction (a) of sulfates (2.3.1).
B. It gives the reactions reaction (a) of potassium (2.3.1).
TESTS
Solution S. Dissolve 10.0 g in 90 mL of carbon dioxide-free water R prepared from distilled
water R, heating gently. Allow to cool and dilute to 100 mL with carbon dioxide-free water R
prepared from distilled water R.
Appearance of solution. Solution S is clear (2.2.1) and colourless (2.2.2, Method II).
Acidity or alkalinity. To 10 mL of solution S add 0.1 mL of bromothymol blue solution R1. Not
more than 0.5 mL of 0.01 M hydrochloric acid or 0.01 M sodium hydroxide is required to change
the colour of the indicator.
Dilute 12.5 mL of solution S to 15 mL with water R.
Calcium (2.4.3) : maximum 200 ppm.
Dilute 5 mL of solution S to 15 mL with distilled water R.
Iron (2.4.9) : maximum 10 ppm, determined on 10 mL of solution S.
Magnesium : maximum 20 ppm.
To 5 mL of solution S add 5 mL of water R, 1 mL of glycerol (85 per cent) R, 0.15 mL of titan
yellow solution R, 0.25 mL of ammonium oxalate solution R and 5 mL of dilute sodium hydroxide
solution R and shake. Any pink colour in the test solution is not more intense than that in
a standard prepared at the same time and in the same manner using a mixture of 1 mL of
magnesium standard solution (10 ppm Mg) R and 9 mL of water R.
Sodium : maximum 0.10 per cent.
Atomic emission spectrometry (2.2.22, Method I).
Test solution. Dissolve 1.00 g of the substance to be examined in water R and dilute to 100.0 mL

Reference solutions. Dissolve in water R 0.50 g of sodium chloride R, previously dried at

100-105 °C for 3 h, and dilute to 1000.0 mL with the same solvent (200 µg of Na per millilitre).
Dilute as required.
Wavelength : 589 nm.
Loss on drying (2.2.32) : maximum 1.0 per cent, determined on 1.000 g by drying in an oven at
130 °C for 4 h.
ASSAY
Dissolve 0.150 g in 40 mL of water R. Add 0.2 mL of 0.1 M hydrochloric acid and 80 mL of
methanol R. Carry out a potentiometric titration (2.2.20), using 0.1 M lead nitrate and as indicator
electrode a lead-selective electrode and as reference electrode a silver-silver chloride electrode.
1 mL of 0.1 M lead nitrate is equivalent to 17.43 mg of K2SO4.
Dissolve 0.610 g in 10 mL of water R. Pass through a column of strongly acidic ion-exchange
resin R at a flow rate of about 4 mL/min. Use 20 g of the resin in 50 mL of water R to prepare the
column of 20 mm internal diameter. Wash with water R (about 80 mL) and check that the pH of
0.05 mL of the eluent is about 6 to 7 using a pH indicator strip R.
Titrate the eluent with 1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20).
1 mL of 1 M sodium hydroxide is equivalent to 87.15 mg of K2SO4.


Related substances : method replaced by a more efficient LC ; specifications updated based
on recent batch data.
Impurities: section added.
XXXX:0635
PRIMAQUINE DIPHOSPHATE
Primaquini diphosphas
C15H27N3O9P2 Mr 455.3
[63-45-6]
DEFINITION
(4RS)-N4-(6-Methoxyquinolin-8-yl)pentane-1,4-diamine bisphosphate.
CHARACTERS
Appearance : orange crystalline powder.
Solubility : soluble in water, practically insoluble in ethanol (96 per cent).
mp : about 200 °C, with decomposition.
IDENTIFICATION
First identification : B, D.
Second identification : A, C, D.
A. Ultraviolet and visible absorption spectrophotometry (2.2.25).
Test solution (a). Dissolve 15 mg in 0.01 M hydrochloric acid and dilute to 100.0 mL with the
same acid.
Test solution (b). Dilute 5.0 mL of test solution (a) to 50.0 mL with 0.01 M hydrochloric acid.
Spectral range : 310-450 nm for test solution (a) ; 215-310 nm for test solution (b).
Absorption maxima : at 332 nm and 415 nm for test solution (a) ; at 225 nm, 265 nm and 282 nm
for test solution (b).
Specific absorbance at the absorption maxima:
– at 332 nm : 45 to 52 for test solution (a) ;
– at 415 nm : 27 to 35 for test solution (a) ;
– at 225 nm : 495 to 515 for test solution (b) ;
– at 265 nm : 335 to 350 for test solution (b) ;
– at 282 nm : 330 to 345 for test solution (b).
B. Infrared absorption spectrophotometry (2.2.24).
Preparation : discs.
Dissolve separately 0.1 g of the substance to be examined and 0.1 g of the reference substance
in 5 mL of water R, add 2 mL of dilute ammonia R2 and 5 mL of methylene chloride R, then
shake. Dry the methylene chloride layer over 0.5 g of anhydrous sodium sulfate R. Prepare a

blank disc using about 0.3 g of potassium bromide R. Apply dropwise to the disc 0.1 mL of the
methylene chloride layer, allowing the methylene chloride to evaporate between applications.
Dry the disc at 50 °C for 2 min.
Comparison : primaquine diphosphate CRS.
C. Thin-layer chromatography (2.2.27). Carry out all operations as rapidly as possible, protected
from light. Prepare the solutions immediately before use.
Test solution. Dissolve 0.20 g of the substance to be examined in 5 mL of water R and dilute to
10 mL with methanol R. Dilute 1 mL of this solution to 10 mL with a mixture of equal volumes of
methanol R and water R.
Reference solution. Dissolve 20 mg of primaquine diphosphate CRS in 5 mL of water R and
dilute to 10 mL with methanol R.
Plate : TLC silica gel GF254 plate R.
Pretreatment : wash the plate with the mobile phase and allow to dry in air.
Mobile phase : concentrated ammonia R, methanol R, methylene chloride R (1:40:60 V/V/V).
Drying : in air.
D. Dissolve 50 mg in 5 mL of water R. Add 2 mL of dilute sodium hydroxide solution R and shake
with 2 quantities, each of 5 mL, of methylene chloride R. The aqueous layer, acidified by
addition of nitric acid R, gives reaction (b) of phosphates (2.3.1).
TESTS
Test solution. Dissolve 50 mg of the substance to be examined in water R and dilute to 5.0 mL
with the same solvent. To 1.0 mL of this solution add 0.2 mL of concentrated ammonia R and
shake with 10.0 mL of the mobile phase. Use the clear lower layer.
Reference solution (a). Dissolve 50 mg of primaquine diphosphate CRS in water R and dilute to
5.0 mL with the same solvent. To 1.0 mL of this solution add 0.2 mL of concentrated ammonia R
and shake with 10.0 mL of the mobile phase. Use the clear lower layer.
Reference solution (b). Dilute 3.0 mL of the test solution to 100.0 mL with the mobile phase.
Reference solution (c). Dilute 1.0 mL of the test solution to 10.0 mL with the mobile phase. Dilute
1.0 mL of this solution to 50.0 mL with the mobile phase.
Column :
– size : l = 0.2 m, Ø = 4.6 mm ;
– stationary phase : silica gel for chromatography R (10 µm).
Mobile phase : concentrated ammonia R, methanol R, hexane R, methylene chloride R
(0.1:10:45:45 V/V/V/V).
Injection : 20 µL.
Run time : at least twice the retention time of primaquine.
– the chromatogram obtained with reference solution (a) shows just before the principal peak a
peak whose area is about 6 per cent of that of the principal peak ;
– resolution : minimum 2.0 between the peak just before the principal peak and the principal peak
in the chromatogram obtained with reference solution (a) ;
– signal-to-noise ratio : minimum 5 for the principal peak in the chromatogram obtained with
reference solution (c).

Limits :
– total : not more than the area of the principal peak in the chromatogram obtained with reference
solution (b) (3.0 per cent) ;
– disregard limit : the area of the principal peak in the chromatogram obtained with reference
solution (c) (0.2 per cent).
Test solution. Dissolve 25.0 mg of the substance to be examined in mobile phase A, using
sonication if necessary, and dilute to 50.0 mL with mobile phase A. Use a freshly prepared solution.
Reference solution (a). Dissolve 5 mg of primaquine for system suitability CRS (containing
impurity A) in mobile phase A and dilute to 10.0 mL with mobile phase A.
Reference solution (b). Dilute 1.0 mL of the test solution to 10.0 mL with mobile phase A. Dilute
1.0 mL of this solution to 100.0 mL with mobile phase A.
Column :
– size : l = 0.15 m, Ø = 4.6 mm ;
– stationary phase : end-capped octylsilyl silica gel for chromatography with polar incorporated
groups R (5 µm)(29) ;
Mobile phase :
– mobile phase A : trifluoroacetic acid R, tetrahydrofuran R, acetonitrile R, water for
chromatography R (0.1/1/9/90 V/V/V/V) ;
– mobile phase B : acetonitrile R ;
0 - 15 100 0
15 - 40 100 → 50 0 → 50
40 - 45 50 50

Injection : 25 µL.
Relative retention with reference to primaquine (retention time = about 13 min) :
impurity A = about 0.86.
– resolution : minimum 2.0 between the peaks due to impurity A and primaquine.
– for each impurity, use the concentration of primaquine in reference solution (b).
Limits :
(29) Symmetry Shield RP 8 is suitable.


Pharmacopoeia.
1. impurity C 2. impurity A 3. primaquine 4. impurity B 5. impurity D
Figure 0635.-1. – Chromatogram for the test for related substances of primaquine diphosphate :
test solution
at 105 °C.
ASSAY
Dissolve 0.2000 g in 40 mL of anhydrous acetic acid R, heating gently. Allow to cool and titrate
with 0.1 M perchloric acid, determining the end-point potentiometrically (2.2.20).
1 mL of 0.1 M perchloric acid is equivalent to 22.77 mg of C15H27N3O9P2.
STORAGE
IMPURITIES
for pharmaceutical use) : A, B, C, D.
A. (4RS)-N1-(6-methoxyquinolin-8-yl)pentane-1,4-diamine (quinocide),
B. 6-methoxy-8-nitroquinoline,

C. 6-methoxyquinolin-8-amine,
D. 2-[(4RS)-4-[(6-methoxyquinolin-8-yl)amino]pentyl]-1H-isoindole-1,3(2H)-dione.


XXXX:0099
SODIUM SULFATE, ANHYDROUS
Natrii sulfas anhydricus
Na2SO4 Mr 142.0
[7757-82-6]
DEFINITION
CHARACTERS
Appearance : white or almost white powder, hygroscopic.
Solubility : freely soluble in water.
IDENTIFICATION
A. It gives the reactions (a) of sulfates (2.3.1).
B. It gives the reactions (a) of sodium (2.3.1).
C. Loss on drying (see Tests).
TESTS
Dilute 5 mL of solution S to 15 mL with water R.
Calcium (2.4.3) : maximum 450 ppm, if intended for use in the manufacture of parenteral
preparations.
Iron (2.4.9) : maximum 90 ppm, if intended for use in the manufacture of parenteral preparations.
Magnesium : maximum 200 ppm, if intended for use in the manufacture of parenteral preparations.
To 10 mL of solution S add 1 mL of glycerol (85 per cent) R, 0.15 mL of titan yellow solution R,
0.25 mL of ammonium oxalate solution R and 5 mL of dilute sodium hydroxide solution R and
shake. Any pink colour in the test solution is not more intense than that in a standard prepared
at the same time in the same manner using a mixture of 5 mL of magnesium standard solution
(10 ppm Mg) R and 5 mL of water R.
at 130 °C.

ASSAY
Dissolve 0.100 g in 40 mL of water R. Add a mixture of 0.2 mL of 0.1 M hydrochloric acid and
80 mL of methanol R. Carry out a potentiometric titration (2.2.20), using 0.1 M lead nitrate and as
indicator electrode a lead-selective electrode and as reference electrode a silver-silver chloride
electrode.
1 mL of 0.1 M lead nitrate is equivalent to 14.20 mg of Na2SO4.
1 mL of 1 M sodium hydroxide is equivalent to 71.00 mg of Na2SO4.
STORAGE
Store in an airtight container.
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture
of parenteral preparations.


XXXX:0100
SODIUM SULFATE DECAHYDRATE
Natrii sulfas decahydricus

Na2SO4,10H2O Mr 322.2
[7727-73-3]
DEFINITION
CHARACTERS
Appearance : white or almost white, crystalline powder or colourless, transparent crystals.
Solubility : freely soluble in water, practically insoluble in ethanol (96 per cent). It partly dissolves
in its own water of crystallisation at about 33 °C.
IDENTIFICATION
A. It gives the reactions reaction (a) of sulfates (2.3.1).
B. It gives the reactions reaction (a) of sodium (2.3.1).
C. Loss on drying (see Tests).
TESTS
Calcium (2.4.3) : maximum 200 ppm, if intended for use in the manufacture of parenteral
preparations.
Iron (2.4.9) : maximum 40 ppm, if intended for use in the manufacture of parenteral preparations.
Magnesium : maximum 100 ppm, if intended for use in the manufacture of parenteral preparations.
To 10 mL of solution S add 1 mL of glycerol (85 per cent) R, 0.15 mL of titan yellow solution R,
0.25 mL of ammonium oxalate solution R and 5 mL of dilute sodium hydroxide solution R and
shake. Any pink colour in the test solution is not more intense than that in a standard prepared
at the same time in the same manner using a mixture of 5 mL of magnesium standard solution
(10 ppm Mg) R and 5 mL of water R.
Loss on drying (2.2.32) : 52.0 per cent to 57.0 per cent, determined on 1.000 g by drying at
30 °C for 1 h, then at 130 °C.

ASSAY
Dissolve 0.250 g in 40 mL of water R. Add a mixture of 0.2 mL of 0.1 M hydrochloric acid and
80 mL of methanol R. Carry out a potentiometric titration (2.2.20), using 0.1 M lead nitrate and as
indicator electrode a lead-selective electrode and as reference electrode a silver-silver chloride
electrode.
1 mL of 0.1 M lead nitrate is equivalent to 14.20 mg of Na2SO4.
1 mL of 1 M sodium hydroxide is equivalent to 161.1 mg of Na2SO4,10H2O.
LABELLING
The label states, where applicable, that the substance is suitable for use in the manufacture
of parenteral preparations.

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© Pharmeuropa 29.

Reference: PA/PH/Exp. MG/T (16) 7 ANP

NOTE ON THE GENERAL CHAPTER

2.2.32. LOSS ON DRYING

PA/PH/Exp. MG/T (16) 7 ANP

Molecular sieve. 1056600.

PA/PH/Exp. MG/T (16) 7 ANP

Reference: PA/PH/Exp. 13H/T (16) 29 ANP

NOTE ON THE GENERAL CHAPTER

2.3.2. IDENTIFICATION OF FATTY OILS BY THIN-LAYER

Figure 2.3.2.-1. – Chromatograms for the identification of fatty oils (method A)

(1) Merck HPTLC Silica gel 60 RP-18 is suitable.

PA/PH/Exp. 13H/T (16) 29 ANP

Figure 2.3.2.-2. – Chromatograms for the identification of fatty oils (method B)

(2) Merck HPTLC Silica gel 60 RP-18 is suitable.

PA/PH/Exp. 13H/T (16) 29 ANP

Reference: PA/PH/Exp. EXT/T (16) 3 ANP

NOTE ON THE GENERAL CHAPTER

5.23. MONOGRAPHS ON HERBAL DRUG EXTRACTS

PA/PH/Exp. EXT/T (16) 3 ANP

Table 5.23.-1. – Classification and principles of production of extracts

PA/PH/Exp. EXT/T (16) 3 ANP

PA/PH/Exp. EXT/T (16) 3 ANP

Reference: PA/PH/Exp. TCM/T (16) 143 ANP

NOTE ON THE MONOGRAPH

PA/PH/Exp. TCM/T (16) 143 ANP

C. Thin-layer chromatography (2.2.27).

Reference solution. Dissolve 1 mg of hederagenin R and 1 mg of oleanolic acid R in 8 mL

Plate : TLC silica gel plate R (2-10 µm)(3).

Mobile phase : glacial acetic acid R, acetone R, toluene R (5:20:80 V/V/V).

Application : 15 µL as bands of 8 mm.

Development : over a path of 6 cm.

(3) Merck HPTLC Si 60 F254 20 ×10 cm is suitable.

PA/PH/Exp. TCM/T (16) 143 ANP

Top of the plate

Reference solution Test solution

PA/PH/Exp. TCM/T (16) 143 ANP

1. oleanolic acid 2. ursolic acid

PA/PH/Exp. TCM/T (16) 143 ANP

A = logarithm of the concentration of oleanolic acid in the test solution, determined

(4) Waters Symmetry C18 is suitable.

PA/PH/Exp. TCM/T (16) 143 ANP

Reference: PA/PH/Exp. 13H/T (16) 66 ANP

NOTE ON THE MONOGRAPH

AMMONIO METHACRYLATE COPOLYMER (TYPE A)

Ammonio methacrylatis copolymerum A

PA/PH/Exp. 13H/T (16) 66 ANP

A. ethyl propenoate (ethyl acrylate),

B. methyl 2-methylpropenoate (methyl methacrylate).

(5) Polygosil 60-7, C18 is suitable.

PA/PH/Exp. 13H/T (16) 66 ANP

Reference: PA/PH/Exp. 13H/T (16) 67 ANP

NOTE ON THE MONOGRAPH

AMMONIO METHACRYLATE COPOLYMER (TYPE B)

Ammonio methacrylatis copolymerum B

PA/PH/Exp. 13H/T (16) 67 ANP

A. ethyl propenoate (ethyl acrylate),

B. methyl 2-methylpropenoate (methyl methacrylate).

PA/PH/Exp. 13H/T (16) 67 ANP

Reference: PA/PH/Exp. 13H/T (16) 31 ANP

NOTE ON THE MONOGRAPH

ARACHIS OIL, HYDROGENATED

Arachidis oleum hydrogenatum